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Sample records for membrane components localize

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-22

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

  3. Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

    PubMed Central

    Mittelmeier, Telsa M.; Thompson, Mark D.; Öztürk, Esra

    2013-01-01

    Like many algae, Chlamydomonas reinhardtii is phototactic, using two anterior flagella to swim toward light optimal for photosynthesis. The flagella are responsive to signals initiated at the photosensory eyespot, which comprises photoreceptors in the plasma membrane and layers of pigment granules in the chloroplast. Phototaxis depends on placement of the eyespot at a specific asymmetric location relative to the flagella, basal bodies, and bundles of two or four highly acetylated microtubules, termed rootlets, which extend from the basal bodies toward the posterior of the cell. Previous work has shown that the eyespot is disassembled prior to cell division, and new eyespots are assembled in daughter cells adjacent to the nascent four-membered rootlet associated with the daughter basal body (D4), but the chronology of these assembly events has not been determined. Here we use immunofluorescence microscopy to follow assembly and acetylation of the D4 rootlet, localization of individual eyespot components in the plasma membrane or chloroplast envelope, and flagellar emergence during and immediately following cell division. We find that the D4 rootlet is assembled before the initiation of eyespot assembly, which occurs within the same time frame as rootlet acetylation and flagellar outgrowth. Photoreceptors in the plasma membrane are correctly localized in eyespot mutant cells lacking pigment granule layers, and chloroplast components of the eyespot assemble in mutant cells in which photoreceptor localization is retarded. The data suggest that plasma membrane and chloroplast components of the eyespot are independently responsive to a cytoskeletal positioning cue. PMID:23873865

  4. Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components.

    PubMed

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

    2016-11-07

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

  5. Common links in the structure and cellular localization of Rhizobium chitolipooligosaccharides and general Rhizobium membrane phospholipid and glycolipid components.

    PubMed

    Cedergren, R A; Lee, J; Ross, K L; Hollingsworth, R I

    1995-04-04

    Several common links between the structural chemistry of the chitolipooligosaccharides of Rhizobium and the general rhizobial membrane lipid and lipopolysaccharide chemistry of these bacteria have been uncovered. Aspects of common chemistry include sulfation, methylation, and the position and extent of fatty acyl chain unsaturation. We find that bacteria which are known to synthesize sulfated chitolipooligosaccharides (such as Rhizobium meliloti strains and the broad-host-range Rhizobium species strain NGR234) also have sulfated lipopolysaccharides. Their common origins of sulfation have been demonstrated by using mutants which are known to be impaired in sulfating their chitolipooligosaccharides. In such cases, there is a corresponding diminution or complete lack of sulfation of the lipopolysaccharides. The structural diversity of the fatty acids observed in the chitolipooligosaccharides is also observed in the other membrane lipids. For instance, the doubly unsaturated fatty acids which are known to be predominant components of R. meliloti chitolipooligosaccharides were also found in the usual phospholipids and glycolipids. Also, the known functionalization of the chitolipooligosaccharides of R. sp. NGR234 by O- and N-methylation was also reflected in the lipopolysaccharide of this organism. The common structural features of chitolipooligosaccharides and membrane components are consistent with a substantial degree of biosynthetic overlap and a large degree of cellular, spatial overlap between these molecules. The latter aspect is clearly demonstrated here since we show that the chitolipooligosaccharides are, in fact, normal membrane components of Rhizobium. This increases the importance of understanding the role of the bacterial cell surface chemistry in the Rhizobium/legume symbiosis and developing a comprehensive understanding of the highly integrated membrane lipid and glycolipid chemistry of Rhizobium.

  6. PINK1 and Parkin control localized translation of respiratory chain component mRNAs on mitochondria outer membrane.

    PubMed

    Gehrke, Stephan; Wu, Zhihao; Klinkenberg, Michael; Sun, Yaping; Auburger, Georg; Guo, Su; Lu, Bingwei

    2015-01-06

    Mitochondria play essential roles in many aspects of biology, and their dysfunction has been linked to diverse diseases. Central to mitochondrial function is oxidative phosphorylation (OXPHOS), accomplished by respiratory chain complexes (RCCs) encoded by nuclear and mitochondrial genomes. How RCC biogenesis is regulated in metazoans is poorly understood. Here we show that Parkinson's disease (PD)-associated genes PINK1 and Parkin direct localized translation of certain nuclear-encoded RCC (nRCC) mRNAs. Translationally repressed nRCC mRNAs are localized in a PINK1/Tom20-dependent manner to mitochondrial outer membrane, where they are derepressed and activated by PINK1/Parkin through displacement of translation repressors, including Pumilio and Glorund/hnRNP-F, a Parkin substrate, and enhanced binding of activators such as eIF4G. Inhibiting the translation repressors rescued nRCC mRNA translation and neuromuscular-degeneration phenotypes of PINK1 mutant, whereas inhibiting eIF4G had opposite effects. Our results reveal previously unknown functions of PINK1/Parkin in RNA metabolism and suggest new approaches to mitochondrial restoration and disease intervention.

  7. Diffusion mediated localization on membrane surfaces

    NASA Technical Reports Server (NTRS)

    Weaver, D. L.

    1982-01-01

    Using the model of a cell membrane of a spherical surface in which membrane components may diffuse, the rate of localization due to trapping under diffusion control has been estimated by computing an analytical expression for the mean trapping time including the possibilities of a trapping probability less than one and/or the establishment of an equilibrium at the trap boundary.

  8. Membrane interactivity of charged local anesthetic derivative and stereoselectivity in membrane interaction of local anesthetic enantiomers

    PubMed Central

    Tsuchiya, Hironori; Mizogami, Maki

    2008-01-01

    With respect to the membrane lipid theory as a molecular mechanism for local anesthetics, two critical subjects, the negligible effects of charged drugs when applied extracellularly and the stereoselective effects of enantiomers, were verified by paying particular attention to membrane components, phospholipids with the anionic property, and cholesterol with several chiral carbons. The membrane interactivities of structurally-different anesthetics were determined by their induced fluidity changes of liposomal membranes. Lidocaine (3.0 μmol/mL) fluidized phosphatidylcholine membranes, but not its quaternary derivative QX-314 (3.0 μmol/mL). Similarly to the mother molecule lidocaine, however, QX-314 fluidized phosphatidylserine-containing nerve cell model membranes and acidic phospholipids-constituting membranes depending on the acidity of membrane lipids. Positively charged local anesthetics are able to act on lipid bilayers by ion-pairing with anionic (acidic) phospholipids. Bupivacaine (0.75 mol/mL) and ropivacaine (0.75 and 1.0 μmol/mL) fluidized nerve cell model membranes with the potency being S(−)-enantiomer < racemate < R(+)-enantiomer (P < 0.01, vs antipode and racemate) and cardiac cell model membranes with the potency being S(−)-ropivacaine < S(−)-bupivacaine < R(+)-bupivacaine (P < 0.01). However, their membrane effects were not different when removing cholesterol from the model membranes. Stereoselectivity is producible by cholesterol which increases the chirality of lipid bilayers and enables to discriminate anesthetic enantiomers. The membrane lipid interaction should be reevaluated as the mode of action of local anesthetics. PMID:22915858

  9. Membrane interactivity of charged local anesthetic derivative and stereoselectivity in membrane interaction of local anesthetic enantiomers.

    PubMed

    Tsuchiya, Hironori; Mizogami, Maki

    2008-01-01

    With respect to the membrane lipid theory as a molecular mechanism for local anesthetics, two critical subjects, the negligible effects of charged drugs when applied extracellularly and the stereoselective effects of enantiomers, were verified by paying particular attention to membrane components, phospholipids with the anionic property, and cholesterol with several chiral carbons. The membrane interactivities of structurally-different anesthetics were determined by their induced fluidity changes of liposomal membranes. Lidocaine (3.0 μmol/mL) fluidized phosphatidylcholine membranes, but not its quaternary derivative QX-314 (3.0 μmol/mL). Similarly to the mother molecule lidocaine, however, QX-314 fluidized phosphatidylserine-containing nerve cell model membranes and acidic phospholipids-constituting membranes depending on the acidity of membrane lipids. Positively charged local anesthetics are able to act on lipid bilayers by ion-pairing with anionic (acidic) phospholipids. Bupivacaine (0.75 mol/mL) and ropivacaine (0.75 and 1.0 μmol/mL) fluidized nerve cell model membranes with the potency being S(-)-enantiomer < racemate < R(+)-enantiomer (P < 0.01, vs antipode and racemate) and cardiac cell model membranes with the potency being S(-)-ropivacaine < S(-)-bupivacaine < R(+)-bupivacaine (P < 0.01). However, their membrane effects were not different when removing cholesterol from the model membranes. Stereoselectivity is producible by cholesterol which increases the chirality of lipid bilayers and enables to discriminate anesthetic enantiomers. The membrane lipid interaction should be reevaluated as the mode of action of local anesthetics.

  10. Dynamics of two-component membranes surrounded by viscoelastic media.

    PubMed

    Komura, Shigeyuki; Yasuda, Kento; Okamoto, Ryuichi

    2015-11-04

    We discuss the dynamics of two-component fluid membranes which are surrounded by viscoelastic media. We assume that membrane-embedded proteins can diffuse laterally and induce a local membrane curvature. The mean squared displacement of a tagged membrane segment is obtained as a generalized Einstein relation. When the elasticity of the surrounding media obeys a power-law behavior in frequency, an anomalous diffusion of the membrane segment is predicted. We also consider the situation where the proteins generate active non-equilibrium forces. The generalized Einstein relation is further modified by an effective temperature that depends on the force dipole energy. The obtained generalized Einstein relations are useful for membrane microrheology experiments.

  11. Polyphophoinositides components of plant nuclear membranes

    SciTech Connect

    Hendrix, K.W.; Boss, W.F.

    1987-04-01

    The polyphosphoinositides, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP/sub 2/), have been shown to be important components in signal transduction in many animal cells. Recently, these lipids have been found to be associated with plasma membrane but not microsomal membrane isolated from fusogenic wild carrot cells; however, in that study the lipids of the nuclear membrane were not analyzed. Since polyphosphoinositides had been shown to be associated with the nuclear membranes as well as the plasma membrane in some animal cells, it was important to determine whether they were associated with plant nuclear membranes as well. Cells were labeled for 18h with (/sup 3/H) inositol and the nuclei were isolated by a modification of the procedure of Saxena et al. Preliminary lipid analyses indicate lower amount of PIP and PIP/sub 2/ in nuclear membranes compared to whole protoplasts. This suggests that the nuclear membranes of carrot cells are not enriched in PIP and PIP/sub 2/; however, the Triton X-100 used during the nuclear isolation procedure may have affected the recovery of the lipids. Experiments are in progress to determine the effects of Triton X-100 on lipid extraction.

  12. Protein-Induced Membrane Curvature Alters Local Membrane Tension

    PubMed Central

    Rangamani, Padmini; Mandadap, Kranthi K.; Oster, George

    2014-01-01

    Adsorption of proteins onto membranes can alter the local membrane curvature. This phenomenon has been observed in biological processes such as endocytosis, tubulation, and vesiculation. However, it is not clear how the local surface properties of the membrane, such as membrane tension, change in response to protein adsorption. In this article, we show that the partial differential equations arising from classical elastic model of lipid membranes, which account for simultaneous changes in shape and membrane tension due to protein adsorption in a local region, cannot be solved for nonaxisymmetric geometries using straightforward numerical techniques; instead, a viscous-elastic formulation is necessary to fully describe the system. Therefore, we develop a viscous-elastic model for inhomogeneous membranes of the Helfrich type. Using the newly available viscous-elastic model, we find that the lipids flow to accommodate changes in membrane curvature during protein adsorption. We show that, at the end of protein adsorption process, the system sustains a residual local tension to balance the difference between the actual mean curvature and the imposed spontaneous curvature. We also show that this change in membrane tension can have a functional impact such as altered response to pulling forces in the presence of proteins. PMID:25099814

  13. [Membrane protein characterization by photoactivatable localization microscopy].

    PubMed

    Huang, Li; Fang, Weihuan; Yu, Ying; Song, Houhui

    2012-11-01

    The on-site labeling and localization tracking of membrane proteins in pathogenic bacteria are tedious work. In order to develop a novel protein labeling technology at super resolution level (nanometer scale) using the photoactivatable localization microscopy (PALM), the chimeric protein of the outer membrane protein A (OmpA) of Mycobacterium tuberculosis and the photoactivatable mEos2m protein were expressed in the non-pathogenic Mycobacterium smegmatis. The recombinant bacteria were fixed on slide, activated by 405 nm laser and subject to PALM imaging to capture photons released by the fusion protein. Meanwhile, colony and cell morphology were visualized under regular fluorescent stereomicroscope and upright fluorescent microscope to characterize fluorescence conversion and protein localization. The fusion proteins formed a "belt"-like structure on cell membrane of M. smegmatis under PALM, providing direct evidence of on-site imaging of membrane proteins. Expression of fusion protein did not compromise the localization properties of OmpA. Thus, mEos2m could be used as a labeling probe to track localizations of non-oligomer oriented membrane proteins. This indicates non-pathogenic M. smegmatis could be served as a model strain to characterize the function and localization of the proteins derived from pathogenic M. tuberculosis. This is the first report using PALM to characterize localization of membrane proteins.

  14. Computational analysis of local membrane properties

    NASA Astrophysics Data System (ADS)

    Gapsys, Vytautas; de Groot, Bert L.; Briones, Rodolfo

    2013-10-01

    In the field of biomolecular simulations, dynamics of phospholipid membranes is of special interest. A number of proteins, including channels, transporters, receptors and short peptides are embedded in lipid bilayers and tightly interact with phospholipids. While the experimental measurements report on the spatial and/or temporal average membrane properties, simulation results are not restricted to the average properties. In the current study, we present a collection of methods for an efficient local membrane property calculation, comprising bilayer thickness, area per lipid, deuterium order parameters, Gaussian and mean curvature. The local membrane property calculation allows for a direct mapping of the membrane features, which subsequently can be used for further analysis and visualization of the processes of interest. The main features of the described methods are highlighted in a number of membrane systems, namely: a pure dimyristoyl-phosphatidyl-choline (DMPC) bilayer, a fusion peptide interacting with a membrane, voltage-dependent anion channel protein embedded in a DMPC bilayer, cholesterol enriched bilayer and a coarse grained simulation of a curved palmitoyl-oleoyl-phosphatidyl-choline lipid membrane. The local membrane property analysis proves to provide an intuitive and detailed view on the observables that are otherwise interpreted as averaged bilayer properties.

  15. Effects of spatial variation in membrane diffusibility and solubility on the lateral transport of membrane components.

    PubMed Central

    Eisinger, J; Halperin, B I

    1986-01-01

    There exist many examples of membrane components (e.g. receptors) accumulating in special domains of cell membranes. We analyze how certain variations in lateral diffusibility and solubility of the membrane would increase the efficiency of transport to these regions. A theorem is derived to show that the mean-time-of capture, tc, for particles diffusing to a trap from an annular region surrounding it, is intermediate to the tc values that correspond to the minimum and maximum diffusion coefficients that obtain in this region. An analytical solution for tc as a function of the gradient of diffusivity surrounding a trap is derived for circular geometry. Since local diffusion coefficients can be increased dramatically by reducing the concentration of intra-membrane particles and/or allowing them to form aggregates, such mechanisms could greatly enhance the diffusion-limited transport of particular membrane components to a trap (e.g. coated pit). If the trap is surrounded by an annular region in which the probe particles' partition function is increased, say, by the local segregation of certain phospholipids, tc is shown to vary inversely with the logarithm of the relative partition function. We provide some conjectural examples to illustrate the magnitude of the effects which heterogeneities in diffusibility and solubility may have in biological membranes. PMID:3756302

  16. Nanoscale Membrane Curvature detected by Polarized Localization Microscopy

    NASA Astrophysics Data System (ADS)

    Kelly, Christopher; Maarouf, Abir; Woodward, Xinxin

    Nanoscale membrane curvature is a necessary component of countless cellular processes. Here we present Polarized Localization Microscopy (PLM), a super-resolution optical imaging technique that enables the detection of nanoscale membrane curvature with order-of-magnitude improvements over comparable optical techniques. PLM combines the advantages of polarized total internal reflection fluorescence microscopy and fluorescence localization microscopy to reveal single-fluorophore locations and orientations without reducing localization precision by point spread function manipulation. PLM resolved nanoscale membrane curvature of a supported lipid bilayer draped over polystyrene nanoparticles on a glass coverslip, thus creating a model membrane with coexisting flat and curved regions and membrane radii of curvature as small as 20 nm. Further, PLM provides single-molecule trajectories and the aggregation of curvature-inducing proteins with super-resolution to reveal the correlated effects of membrane curvature, dynamics, and molecular sorting. For example, cholera toxin subunit B has been observed to induce nanoscale membrane budding and concentrate at the bud neck. PLM reveals a previously hidden and critical information of membrane topology.

  17. Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy.

    PubMed

    Kowalik, Magdalena K; Rekawiecki, Robert; Kotwica, Jan

    2014-11-01

    The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5-6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Separation of components in lipid membranes induced by shape transformation

    NASA Astrophysics Data System (ADS)

    Góźdź, W. T.; Bobrovska, N.; Ciach, A.

    2012-07-01

    Vesicles composed of a two component membrane with each component characterized by different spontaneous curvature are investigated by minimization of the free energy consisting of Helfrich elastic energy and entropy of mixing. The results show that mixing and demixing of membrane components can be induced by elongating a vesicle or changing its volume, if one of the components forms a complex with macromolecules on the outer monolayer. The influence of elastic coefficients on the separation of components is also examined.

  19. Interactions of some local anesthetics and alcohols with membranes.

    PubMed

    Frangopol, P T.; Mihăilescu, D

    2001-09-01

    A review of the results obtained by our group in the last decade regarding the interactions of procaine, lidocaine, dibucaine and tetracaine with membranes is presented in the context of the literature data. The action upon membranes, in first approximation monomolecular film of stearic acid spread at the air/water interface used as a membrane model, the modification of biomembrane structure and function using diffraction methods, lipid phase transition, fluidity of lipids and proteins, membrane expansion and platelet aggregation were studied. The thermodynamic knowledge of membrane-alcohol interactions improved by using highly sensitive calorimetric techniques are briefly reported. One of the main conclusions is that the physical state of a monolayer model membrane was the result of competitive interactions between film-film and film-substrate interactions. It was taken into account that local anesthetics, such as lidocaine, carbisocaine, mesocaine, showed changes in the bilayer structure, reflected in macroscopic mechanical properties. This restructuring of the lipid bilayer has a significant influence on the operation of functional subunits, e.g. ionic channels formed by gramicidin. The results support the concept of non-specific interactions of local anesthetics with lipid bilayers. The theoretical modeling of the interactions of local anesthetics is closely compared with experimental data. Our new theory of relaxation for these interactions is using a non-archimedean formalism based on a process resulting from superpositions of different component processes which take place at different scales of time.

  20. Analysis of truss, beam, frame, and membrane components. [composite structures

    NASA Technical Reports Server (NTRS)

    Knoell, A. C.; Robinson, E. Y.

    1975-01-01

    Truss components are considered, taking into account composite truss structures, truss analysis, column members, and truss joints. Beam components are discussed, giving attention to composite beams, laminated beams, and sandwich beams. Composite frame components and composite membrane components are examined. A description is given of examples of flat membrane components and examples of curved membrane elements. It is pointed out that composite structural design and analysis is a highly interactive, iterative procedure which does not lend itself readily to characterization by design or analysis function only.-

  1. Mutual Interactions between Aquaporins and Membrane Components

    PubMed Central

    Martínez-Ballesta, Maria del Carmen; Carvajal, Micaela

    2016-01-01

    In recent years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new forms of regulation of aquaporins described, based on these associations. Heterotetramerizations of aquaporin isoforms are considered as novel regulatory mechanisms for plasma membrane (PIPs) and tonoplast (TIPs) proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein–protein interaction is an extensive theme that is difficult to tackle and new methodologies are being used to study the physical interactions involved. Bimolecular fluorescence complementation and the identification of cross-linked peptides based on tandem mass spectra, that are complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, are discussed in this review. The chemical composition and the physical characteristics of the lipid bilayer also influence many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the positions of the lipids around aquaporins could define their activity, thereby altering the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is provided. Finally, it is described how the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances. PMID:27625676

  2. Mutual Interactions between Aquaporins and Membrane Components.

    PubMed

    Martínez-Ballesta, Maria Del Carmen; Carvajal, Micaela

    2016-01-01

    In recent years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new forms of regulation of aquaporins described, based on these associations. Heterotetramerizations of aquaporin isoforms are considered as novel regulatory mechanisms for plasma membrane (PIPs) and tonoplast (TIPs) proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein-protein interaction is an extensive theme that is difficult to tackle and new methodologies are being used to study the physical interactions involved. Bimolecular fluorescence complementation and the identification of cross-linked peptides based on tandem mass spectra, that are complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, are discussed in this review. The chemical composition and the physical characteristics of the lipid bilayer also influence many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the positions of the lipids around aquaporins could define their activity, thereby altering the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is provided. Finally, it is described how the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances.

  3. Regulation of photosynthetic membrane components in cyanobacteria

    SciTech Connect

    Sherman, L.A.

    1991-01-01

    The goals of this proposal were two-fold: (1) to analyze the impact of mutations in the Mn-stabilizing protein (MSP) on O{sub 2}- evolution; and (2) to analyze the effect of iron deficiency on membrane assembly in cyanobacteria. We have made important progress in both projects, and I will discuss each of them in turn. The mutations in the psbO gene were performed in the transformable and photoheterotrophic cyanobacterium Synechocystis sp. PCC6803; this strain allows PSII mutations to be propagated under nonphotosynthetic conditions. The research with iron deficiency was performed in the cyanobacterium Synechococcus sp. PCC7942, which is transformable and which has been used previously for all our nutritional-deficiency research. 5 figs.

  4. Depot effect of bioactive components in experimental membrane filtrations

    NASA Astrophysics Data System (ADS)

    Mitev, D.; Peshev, D.; Peev, G.; Peeva, L.

    2017-01-01

    Depot effects were found to be accompanying phenomena of membrane separation processes. Accumulation of target species in the membrane matrix during feasibility tests can hamper proper conclusions or compromise the filtration results. Therefore, we investigated the effects of delayed membrane release of chlorogenic acid and caffeine, considered as key compounds of interest in spent coffee products’ recovery treatment. Permeate fluxes and key components release were studied in course of 24 hours via nanofiltration of pure solvent, both immediately after the mock solution filtration and after idle stay. Conclusions are drawn and recommendations advised for proper analysis of experimental data on membrane screening.

  5. The transport along membrane nanotubes driven by the spontaneous curvature of membrane components.

    PubMed

    Kabaso, Doron; Bobrovska, Nataliya; Góźdź, Wojciech; Gongadze, Ekaterina; Kralj-Iglič, Veronika; Zorec, Robert; Iglič, Aleš

    2012-10-01

    Intercellular membrane nanotubes (ICNs) serve as a very specific transport system between neighboring cells. The underlying mechanisms responsible for the transport of membrane components and vesicular dilations along the ICNs are not clearly understood. The present study investigated the spatial distribution of anisotropic membrane components of tubular shapes and isotropic membrane components of spherical shapes. Experimental results revealed the preferential distribution of CTB (cholera toxin B)-GM1 complexes mainly on the spherical cell membrane, and cholesterol-sphingomyelin at the membrane leading edge and ICNs. In agreement with previous studies, we here propose that the spatial distribution of CTB-GM1 complexes and cholesterol-sphingomyelin rafts were due to their isotropic and anisotropic shapes, respectively. To elucidate the relationship between a membrane component shape and its spatial distribution, a two-component computational model was constructed. The minimization of the membrane bending (free) energy revealed the enrichment of the anisotropic component along the ICN and the isotropic component in the parent cell membrane, which was due to the curvature mismatch between the ICN curvature and the spontaneous curvature of the isotropic component. The equations of motion, derived from the differentiation of the membrane free energy, revealed a curvature-dependent flux of the isotropic component and a curvature-dependent force exerted on a vesicular dilation along the ICN. Finally, the effects of possible changes in the orientational ordering of the anisotropic component attendant to the transport of the vesicular dilation were discussed with connection to the propagation of electrical and chemical signals. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Cyclical components of local rainfall data

    NASA Astrophysics Data System (ADS)

    Mentz, R. P.; D'Urso, M. A.; Jarma, N. M.; Mentz, G. B.

    2000-02-01

    This paper reports on the use of a comparatively simple statistical methodology to study local short time series rainfall data. The objective is to help in agricultural planning, by diminishing the risks associated with some uncertainties affecting this business activity.The analysis starts by assuming a model of unobservable components, trend, cycle, seasonal and irregular, that is well known in many areas of application. When series are in the realm of business and economics, the statistical methods popularized by the US Census Bureau US National Bureau of Economic Research are used for seasonal and cyclical estimation, respectively. The flexibility of these methods makes them good candidates to be applied in the meteorological context, and this is done in this paper for a selection of monthly rainfall time series.Use of the results to help in analysing and forecasting cyclical components is emphasized. The results are interesting. An agricultural entrepreneur, or a group of them located in a single geographical region, will profit by systematically collecting information (monthly in our work) about rainfall, and adopting the scheme of analysis described in this paper.

  7. Vapour and acid components separation from gases by membranes principles and engineering approach to membranes development

    NASA Astrophysics Data System (ADS)

    Kagramanov, G. G.; Storojuk, I. P.; Farnosova, E. N.

    2016-09-01

    The modern commercially available polymer membranes and membrane modules for purification of gases, containing acid components, simultaneously with dehumidification of treated gas streams, were developed and commercialized in the very end of XXth century. The membranes basic properties - selectivity (separation factor) and permeation flow rates - are relatively far from satisfying the growing and modern-scale industrial need in purification technologies and corresponding equipments. The attempt to formulate the basic principles, scientific and engineering approaches to the development of prospective membranes for the purification of gases, especially such as natural and oil gases, from acid components, simultaneously with drying them, was being made. For this purpose the influence of various factors - polymer nature, membrane type, structure, geometrical and mass-transfer characteristics, etc. - were studied and analyzed in order to formulate the basic principles and demands for development of membranes, capable to withstand successfully the sever conditions of exploitation.

  8. Menin localization in cell membrane compartment

    PubMed Central

    He, Xin; Wang, Lei; Yan, Jizhou; Yuan, Chaoxing; Witze, Eric S.; Hua, Xianxin

    2016-01-01

    ABSTRACT Menin is encoded by the MEN1 gene, which is mutated in an inherited human syndrome, multiple endocrine neoplasia type 1(MEN1). Menin is primarily nuclear protein, acting as a tumor suppressor in endocrine organs, but as an oncogenic factor in the mixed lineage leukemia, in a tissue-specific manner. Recently, the crystal structures of menin with different binding partners reveal menin as a key scaffold protein that functionally interacts with various partners to regulate gene transcription in the nucleus. However, outside the nucleus, menin also regulates multiple signaling pathways that traverse the cell surface membrane. The precise nature regarding to how menin associates with the membrane fraction is poorly understood. Here we show that a small fraction of menin associates with the cell membrane fraction likely via serine palmitoylation. Moreover, the majority of the membrane-associated menin may reside inside membrane vesicles, as menin is protected from trypsin-mediated proteolysis, but disruption of the membrane fraction using detergent abolishes the detection. Consistently, cellular staining for menin also reveals the distribution of menin in the cell membrane and the punctate-like cell organelles. Our findings suggest that part of intracellular menin associates with the cell membrane peripherally as well as resides within the membrane vesicles. PMID:26560942

  9. The extracellular matrix protein WARP is a novel component of a distinct subset of basement membranes.

    PubMed

    Allen, Justin M; Brachvogel, Bent; Farlie, Peter G; Fitzgerald, Jamie; Bateman, John F

    2008-05-01

    WARP is a recently described member of the von Willebrand factor A domain superfamily of extracellular matrix proteins, and is encoded by the Vwa1 gene. We have previously shown that WARP is a multimeric component of the chondrocyte pericellular matrix in articular cartilage and intervertebral disc, where it interacts with the basement membrane heparan sulfate proteoglycan perlecan. However, the tissue-specific expression of WARP in non-cartilaginous tissues and its localization in the extracellular matrix of other perlecan-containing tissues have not been analyzed in detail. To visualize WARP-expressing cells, we generated a reporter gene knock-in mouse by targeted replacement of the Vwa1 gene with beta-galactosidase. Analysis of reporter gene expression and WARP protein localization by immunostaining demonstrates that WARP is a component of a limited number of distinct basement membranes. WARP is expressed in the vasculature of neural tissues and in basement membrane structures of the peripheral nervous system. Furthermore, WARP is also expressed in the apical ectodermal ridge of developing limb buds, and in skeletal and cardiac muscle. These findings are the first evidence for WARP expression in non-cartilaginous tissues, and the identification of WARP as a component of a limited range of specialized basement membranes provides further evidence for the heterogeneous composition of basement membranes between different tissues.

  10. Physical aspects of heterogeneities in multi-component lipid membranes.

    PubMed

    Komura, Shigeyuki; Andelman, David

    2014-06-01

    Ever since the raft model for biomembranes has been proposed, the traditional view of biomembranes based on the fluid-mosaic model has been altered. In the raft model, dynamical heterogeneities in multi-component lipid bilayers play an essential role. Focusing on the lateral phase separation of biomembranes and vesicles, we review some of the most relevant research conducted over the last decade. We mainly refer to those experimental works that are based on physical chemistry approach, and to theoretical explanations given in terms of soft matter physics. In the first part, we describe the phase behavior and the conformation of multi-component lipid bilayers. After formulating the hydrodynamics of fluid membranes in the presence of the surrounding solvent, we discuss the domain growth-law and decay rate of concentration fluctuations. Finally, we review several attempts to describe membrane rafts as two-dimensional microemulsion.

  11. Localization of Sublexical Speech Perception Components

    ERIC Educational Resources Information Center

    Turkeltaub, Peter E.; Coslett, H. Branch

    2010-01-01

    Models of speech perception are in general agreement with respect to the major cortical regions involved, but lack precision with regard to localization and lateralization of processing units. To refine these models we conducted two Activation Likelihood Estimation (ALE) meta-analyses of the neuroimaging literature on sublexical speech perception.…

  12. Localization of Sublexical Speech Perception Components

    ERIC Educational Resources Information Center

    Turkeltaub, Peter E.; Coslett, H. Branch

    2010-01-01

    Models of speech perception are in general agreement with respect to the major cortical regions involved, but lack precision with regard to localization and lateralization of processing units. To refine these models we conducted two Activation Likelihood Estimation (ALE) meta-analyses of the neuroimaging literature on sublexical speech perception.…

  13. Exploration of shape variation using localized components analysis.

    PubMed

    Alcantara, Dan A; Carmichael, Owen; Harcourt-Smith, Will; Sterner, Kirstin; Frost, Stephen R; Dutton, Rebecca; Thompson, Paul; Delson, Eric; Amenta, Nina

    2009-08-01

    Localized Components Analysis (LoCA) is a new method for describing surface shape variation in an ensemble of objects using a linear subspace of spatially localized shape components. In contrast to earlier methods, LoCA optimizes explicitly for localized components and allows a flexible trade-off between localized and concise representations, and the formulation of locality is flexible enough to incorporate properties such as symmetry. This paper demonstrates that LoCA can provide intuitive presentations of shape differences associated with sex, disease state, and species in a broad range of biomedical specimens, including human brain regions and monkey crania.

  14. Regulation of ROCKII membrane localization through its C-terminus.

    PubMed

    Kher, Swapnil S; Worthylake, Rebecca A

    2011-12-10

    RhoA activated kinases (ROCKs) are potent effectors of RhoA signaling for regulation of the cytoskeleton. ROCKs have been shown to be localized to several different subcellular locations, suggesting that its localization is context specific and regulated. However, the signaling mechanisms that control ROCK localization have not been clearly described. In this study we measured ROCKII localization following stimulation with the chemokine CXCL12 or adhesion to collagen 1. Strikingly, each of these extracellular signals targeted ROCKII to membrane protrusions. We further determined that both RhoA and PI3-kinase signaling are required for these stimuli to induce efficient membrane localization. Furthermore, we used a mutational approach to show that two separate domains predicted to respond to these localization signals, the Rho Binding Domain (RBD) and the Pleckstrin Homology domain (PH). Unexpectedly, we found that these two domains work synergistically to lead to membrane localization. This suggests a novel mechanism for controlling ROCKII localization at the membrane, in which the ROCKII C-terminus acts as a coincidence detector for spatial regulatory signals. In other words, efficient membrane targeting requires the ROCKII RBD to receive the RhoA signal and the PH domain to receive the phospholipid signal. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Localization of Sublexical Speech Perception Components

    PubMed Central

    Turkeltaub, Peter E; Coslett, H. Branch

    2010-01-01

    Models of speech perception are in general agreement with respect to the major cortical regions involved, but lack precision with regard to localization and lateralization of processing units. To refine these models we conducted two Activation Likelihood Estimation (ALE) meta-analyses of the neuroimaging literature on sublexical speech perception. Based on foci reported in 23 fMRI experiments, we identified significant activation likelihoods in left and right superior temporal cortex and the left posterior middle frontal gyrus. Subanalyses examining phonetic and phonological processes revealed only left mid-posterior superior temporal sulcus activation likelihood. A lateralization analysis demonstrated temporal lobe left lateralization in terms of magnitude, extent, and consistency of activity. Experiments requiring explicit attention to phonology drove this lateralization. An ALE analysis of eight fMRI studies on categorical phoneme perception revealed significant activation likelihood in the left supramarginal gyrus and angular gyrus. These results are consistent with a speech processing network in which the bilateral superior temporal cortices perform acoustic analysis of speech and nonspeech auditory stimuli, the left mid-posterior superior temporal sulcus performs phonetic and phonological analysis, and the left inferior parietal lobule is involved in detection of differences between phoneme categories. These results modify current speech perception models in three ways: 1) specifying the most likely locations of dorsal stream processing units, 2) clarifying that phonetic and phonological superior temporal sulcus processing is left lateralized and localized to the mid-posterior portion, and 3) suggesting that both the supramarginal gyrus and angular gyrus may be involved in phoneme discrimination. PMID:20413149

  16. Tissue inhibitor of metalloproteinases-3 is a component of Bruch's membrane of the eye.

    PubMed Central

    Fariss, R. N.; Apte, S. S.; Olsen, B. R.; Iwata, K.; Milam, A. H.

    1997-01-01

    Mutations in tissue inhibitor of metalloproteinases (TIMP)-3 are found in some patients with Sorsby's fundus dystrophy, a retinal degeneration characterized by abnormal deposits in Bruch's membrane and choroidal neovascularization. The purpose of this study was to localize TIMP-3 in the retina/choroid of normal human and animal eyes. Immunolabeling was performed on unfixed and fixed sections of human eyes aged 24 to 85 years and unfixed sections of baboon, chicken, cow, pig, and rat eyes using a monoclonal antibody against a human TIMP-3 synthetic peptide. The antibody produced strong immunolabeling of Bruch's membrane and drusen and weak labeling of retina blood vessels in unfixed human and baboon eyes. Unfixed chicken, cow, pig, and rat tissues showed no reactivity. After antigen retrieval, all fixed human eyes showed specific labeling of Bruch's membrane and drusen, which was strongest in eyes from elderly donors. The results indicate that TIMP-3 is an extracellular matrix component of Bruch's membrane. Thus, abnormal local function of TIMP-3 may lead to the characteristic Bruch's membrane deposits and choroidal neovascularization found in Sorsby's fundus dystrophy. Specific labeling of drusen raises the possibility that altered TIMP-3-mediated matrix remodeling may contribute to age-related degenerative changes in Bruch's membrane. Images Figure 1 Figure 2 PMID:9006347

  17. Subcellular localization of mammalian type II membrane proteins.

    PubMed

    Aturaliya, Rajith N; Fink, J Lynn; Davis, Melissa J; Teasdale, Melvena S; Hanson, Kelly A; Miranda, Kevin C; Forrest, Alistair R R; Grimmond, Sean M; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Teasdale, Rohan D

    2006-05-01

    Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

  18. Do local anesthetics interact preferentially with membrane lipid rafts? Comparative interactivities with raft-like membranes.

    PubMed

    Tsuchiya, Hironori; Ueno, Takahiro; Mizogami, Maki; Takakura, Ko

    2010-08-01

    Membranous lipid bilayers have been reconsidered as the site of action of local anesthetics (LAs). Recent understanding of biomembranes indicates the existence of lipid raft microdomains enriched in cholesterol and sphingolipids as potential platforms for channels and receptors. Based on the hypothesis that LAs may interact preferentially with lipid rafts over non-raft membranes, we compared their effects on raft model membranes and cardiolipin-containing biomimetic membranes. Liposomes were prepared with phospholipids, sphingomyelin, cerebroside, and cholesterol to have compositions corresponding to lipid rafts and cardiomyocyte mitochondrial membranes. After reacting LAs (50-200 microM) with the membrane preparations, their interactivities were determined by measuring fluorescence polarization with 1,6-diphenyl-1,3,5-hexatriene. Although bupivacaine and lidocaine acted on different raft-like liquid-ordered membranes to reduce polarization values, their effects on biomimetic less ordered membranes were much greater. LAs interacted with biomimetic membranes with the potency being R(+)-bupivacaine > racemic bupivacaine > S(-)-bupivacaine > ropivacaine > lidocaine > prilocaine, which is consistent with the rank order of pharmacotoxicological potency. However, raft model membranes showed neither structure-dependence nor stereoselectivity. The relevance of membrane lipid rafts to LAs is questionable at least in their effects on raft-like liquid-ordered membranes.

  19. Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

    PubMed Central

    Phillips, John H.

    1973-01-01

    Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg2+-dependent and is stimulated by Mn2+ and F− ions. The initial reaction is rapid, with a broad pH profile and a `transition' temperature for its activation energy at 27°C. The apparent Km for ATP is 5μm. ATP, N-ethylmaleimide, Cu2+ ions and NaIO4 are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2–3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo. PMID:4360713

  20. Membrane Vesicles: an Overlooked Component of the Matrices of Biofilms

    PubMed Central

    Schooling, Sarah R.; Beveridge, Terry J.

    2006-01-01

    The matrix helps define the architecture and infrastructure of biofilms and also contributes to their resilient nature. Although many studies continue to define the properties of both gram-positive and gram-negative bacterial biofilms, there is still much to learn, especially about how structural characteristics help bridge the gap between the chemistry and physical aspects of the matrix. Here, we show that membrane vesicles (MVs), structures derived from the outer membrane of gram-negative bacteria, are a common particulate feature of the matrix of Pseudomonas aeruginosa biofilms. Biofilms grown using different model systems and growth conditions were shown to contain MVs when thin sectioned for transmission electron microscopy, and mechanically disrupted biofilms revealed MVs in association with intercellular material. MVs were also isolated from biofilms by employing techniques for matrix isolation and a modified MV isolation protocol. Together these observations verified the presence and frequency of MVs and indicated that MVs were a definite component of the matrix. Characterization of planktonic and biofilm-derived MVs revealed quantitative and qualitative differences between the two and indicated functional roles, such as proteolytic activity and binding of antibiotics. The ubiquity of MVs was supported by observations of biofilms from a variety of natural environments outside the laboratory and established MVs as common biofilm constituents. MVs appear to be important and relatively unacknowledged particulate components of the matrix of gram-negative or mixed bacterial biofilms. PMID:16885463

  1. Localized translation near the mitochondrial outer membrane: An update.

    PubMed

    Lesnik, Chen; Golani-Armon, Adi; Arava, Yoav

    2015-01-01

    Local synthesis of proteins near their activity site has been demonstrated in many biological systems, and has diverse contributions to cellular functions. Studies in recent years have revealed that hundreds of mitochondria-destined proteins are synthesized by cytosolic ribosomes near the mitochondrial outer membrane, indicating that localized translation also occurs at this cellular locus. Furthermore, in the last year central factors that are involved in this process were identified in yeast, Drosophila, and human cells. Herein we review the experimental evidence for localized translation on the cytosolic side of the mitochondrial outer membrane; in addition, we describe the factors that are involved in this process and discuss the conservation of this mechanism among various species. We also describe the relationship between localized translation and import into the mitochondria and suggest avenues of study that look beyond cotranslational import. Finally we discuss future challenges in characterizing the mechanisms for localized translation and its physiological significance.

  2. Stereostructure-based differences in the interactions of cardiotoxic local anesthetics with cholesterol-containing biomimetic membranes.

    PubMed

    Tsuchiya, Hironori; Ueno, Takahiro; Mizogami, Maki

    2011-06-01

    Amide-type pipecoloxylidide local anesthetics, bupivacaine, and ropivacaine, show cardiotoxic effects with the potency depending on stereostructures. Cardiotoxic drugs not only bind to cardiomyocyte membrane channels to block them but also modify the physicochemical property of membrane lipid bilayers in which channels are embedded. The opposite configurations allow enantiomers to be discriminated by their enantiospecific interactions with another chiral molecule in membranes. We compared the interactions of local anesthetic stereoisomers with biomimetic membranes consisting of chiral lipid components, the differences of which might be indicative of the drug design for reducing cardiotoxicity. Fluorescent probe-labeled biomimetic membranes were prepared with cardiolipin and cholesterol of varying compositions and different phospholipids. Local anesthetics were reacted with the membrane preparations at a cardiotoxically relevant concentration of 200 μM. The potencies to interact with biomimetic membranes and change their fluidity were compared by measuring fluorescence polarization. All local anesthetics acted on lipid bilayers to increase membrane fluidity. Chiral cardiolipin was ineffective in discriminating S(-)-enantiomers from their antipodes. On the other hand, cholesterol produced the enantiospecific membrane interactions of bupivacaine and ropivacaine with increasing its composition in membranes. In 40 mol% and more cholesterol-containing membranes, the membrane-interacting potency was S(-)-bupivacainemembranes in increasing order of intensity. The rank order of membrane interactivity agreed with that of known cardiotoxicity. The stereoselective membrane interactions determined by cholesterol with higher chirality appears to be associated with the

  3. Localization of integral membrane peptidylglycine alpha-amidating monooxygenase in neuroendocrine cells.

    PubMed

    Milgram, S L; Kho, S T; Martin, G V; Mains, R E; Eipper, B A

    1997-03-01

    Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.

  4. Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

    PubMed Central

    1985-01-01

    We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes. PMID:3988805

  5. Polycyclic Aromatic Hydrocarbons as Plausible Prebiotic Membrane Components

    NASA Astrophysics Data System (ADS)

    Groen, Joost; Deamer, David W.; Kros, Alexander; Ehrenfreund, Pascale

    2012-08-01

    Aromatic molecules delivered to the young Earth during the heavy bombardment phase in the early history of our solar system were likely to be among the most abundant and stable organic compounds available. The Aromatic World hypothesis suggests that aromatic molecules might function as container elements, energy transduction elements and templating genetic components for early life forms. To investigate the possible role of aromatic molecules as container elements, we incorporated different polycyclic aromatic hydrocarbons (PAH) in the membranes of fatty acid vesicles. The goal was to determine whether PAH could function as a stabilizing agent, similar to the role that cholesterol plays in membranes today. We studied vesicle size distribution, critical vesicle concentration and permeability of the bilayers using C6-C10 fatty acids mixed with amphiphilic PAH derivatives such as 1-hydroxypyrene, 9-anthracene carboxylic acid and 1,4 chrysene quinone. Dynamic Light Scattering (DLS) spectroscopy was used to measure the size distribution of vesicles and incorporation of PAH species was established by phase-contrast and epifluorescence microscopy. We employed conductimetric titration to determine the minimal concentration at which fatty acids could form stable vesicles in the presence of PAHs. We found that oxidized PAH derivatives can be incorporated into decanoic acid (DA) vesicle bilayers in mole ratios up to 1:10 (PAH:DA). Vesicle size distribution and critical vesicle concentration were largely unaffected by PAH incorporation, but 1-hydroxypyrene and 9-anthracene carboxylic acid lowered the permeability of fatty acid bilayers to small solutes up to 4-fold. These data represent the first indication of a cholesterol-like stabilizing effect of oxidized PAH derivatives in a simulated prebiotic membrane.

  6. Polycyclic aromatic hydrocarbons as plausible prebiotic membrane components.

    PubMed

    Groen, Joost; Deamer, David W; Kros, Alexander; Ehrenfreund, Pascale

    2012-08-01

    Aromatic molecules delivered to the young Earth during the heavy bombardment phase in the early history of our solar system were likely to be among the most abundant and stable organic compounds available. The Aromatic World hypothesis suggests that aromatic molecules might function as container elements, energy transduction elements and templating genetic components for early life forms. To investigate the possible role of aromatic molecules as container elements, we incorporated different polycyclic aromatic hydrocarbons (PAH) in the membranes of fatty acid vesicles. The goal was to determine whether PAH could function as a stabilizing agent, similar to the role that cholesterol plays in membranes today. We studied vesicle size distribution, critical vesicle concentration and permeability of the bilayers using C(6)-C(10) fatty acids mixed with amphiphilic PAH derivatives such as 1-hydroxypyrene, 9-anthracene carboxylic acid and 1,4 chrysene quinone. Dynamic Light Scattering (DLS) spectroscopy was used to measure the size distribution of vesicles and incorporation of PAH species was established by phase-contrast and epifluorescence microscopy. We employed conductimetric titration to determine the minimal concentration at which fatty acids could form stable vesicles in the presence of PAHs. We found that oxidized PAH derivatives can be incorporated into decanoic acid (DA) vesicle bilayers in mole ratios up to 1:10 (PAH:DA). Vesicle size distribution and critical vesicle concentration were largely unaffected by PAH incorporation, but 1-hydroxypyrene and 9-anthracene carboxylic acid lowered the permeability of fatty acid bilayers to small solutes up to 4-fold. These data represent the first indication of a cholesterol-like stabilizing effect of oxidized PAH derivatives in a simulated prebiotic membrane.

  7. Assessment of sulfur mustard interaction with basement membrane components

    SciTech Connect

    Zhang, Z.; Peters, B.P.; Monteiro-Rivier, N.A.

    1995-08-01

    Bis-2-chloroethyl sulfide (sulfur mustard, RD) is a bifunctional alkylating agent which causes severe vesication characterized by slow wound healing. Our previous studies have shown that the vesicant RD disrupts the epidermal-dermal junction at the lamina lucida of the basement membrane. The purpose of this study was to examine whether RD directly modifies basement membrane components (BMCs), and to evaluate the effect of RD on the cell adhesive activity of BMCs. EHS laminin was incubated with (14C)HRD, and extracted by gel filtration. Analysis of the (14C)HRD-conjugated laminin fraction by a reduced sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SD S-PAGE) revealed the incorporation of radioactivity into both laminin subunits and a laminin trimer resistant to dissociation in reduced SDS-PAGE sample buffer, suggesting direct alkylation and cross-linking of EHS laminin by (14C)HD. Normal human foreskin epidermal keratinocytes were biosynthetically labeled with (35S)cysteine. (35S)-labeled laminin isoforms, Ae.Ble.B2e. laminin and K.Ble.B2e. laminin (using the nomenclature of Engel), fibronectin, and heparan sulfate proteoglycan were isolated by irnmunoprecipitation from the cell culture medium, treated with RD or ethanol as control, and then analyzed by SDS-PAGE.

  8. Progesterone receptor membrane component-1 regulates hepcidin biosynthesis.

    PubMed

    Li, Xiang; Rhee, David K; Malhotra, Rajeev; Mayeur, Claire; Hurst, Liam A; Ager, Emily; Shelton, Georgia; Kramer, Yael; McCulloh, David; Keefe, David; Bloch, Kenneth D; Bloch, Donald B; Peterson, Randall T

    2016-01-01

    Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.

  9. Progesterone receptor membrane component-1 regulates hepcidin biosynthesis

    PubMed Central

    Li, Xiang; Rhee, David K.; Malhotra, Rajeev; Mayeur, Claire; Hurst, Liam A.; Ager, Emily; Shelton, Georgia; Kramer, Yael; McCulloh, David; Keefe, David; Bloch, Kenneth D.; Bloch, Donald B.; Peterson, Randall T.

    2015-01-01

    Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism. PMID:26657863

  10. Differential Localization of the Streptococcal Accessory Sec Components and Implications for Substrate Export

    PubMed Central

    Yen, Yihfen T.; Cameron, Todd A.; Bensing, Barbara A.; Seepersaud, Ravin; Zambryski, Patricia C.

    2013-01-01

    The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation. PMID:23204472

  11. Both phototropin 1 and 2 localize on the chloroplast outer membrane with distinct localization activity.

    PubMed

    Kong, Sam-Geun; Suetsugu, Noriyuki; Kikuchi, Shingo; Nakai, Masato; Nagatani, Akira; Wada, Masamitsu

    2013-01-01

    Chloroplasts change their position to adapt cellular activities to fluctuating environmental light conditions. Phototropins (phot1 and phot2 in Arabidopsis) are plant-specific blue light photoreceptors that perceive changes in light intensity and direction, and mediate actin-based chloroplast photorelocation movements. Both phot1 and phot2 regulate the chloroplast accumulation response, while phot2 is mostly responsible for the regulation of the avoidance response. Although it has been widely accepted that distinct intracellular localizations of phototropins are implicated in the specificity, the mechanism underlying the phot2-specific avoidance response has remained elusive. In this study, we examined the relationship of the phot2 localization pattern to the chloroplast photorelocation movement. First, the fusion of a nuclear localization signal with phot2, which effectively reduced the amount of phot2 in the cytoplasm, retained the activity for both the accumulation and avoidance responses, indicating that membrane-localized phot2 but not cytoplasmic phot2 is functional to mediate the responses. Importantly, some fractions of phot2, and of phot1 to a lesser extent, were localized on the chloroplast outer membrane. Moreover, the deletion of the C-terminal region of phot2, which was previously shown to be defective in blue light-induced Golgi localization and avoidance response, affected the localization pattern on the chloroplast outer membrane. Taken together, these results suggest that dynamic phot2 trafficking from the plasma membrane to the Golgi apparatus and the chloroplast outer membrane might be involved in the avoidance response.

  12. Lattice Independent Component Analysis for Mobile Robot Localization

    NASA Astrophysics Data System (ADS)

    Villaverde, Ivan; Fernandez-Gauna, Borja; Zulueta, Ekaitz

    This paper introduces an approach to appearance based mobile robot localization using Lattice Independent Component Analysis (LICA). The Endmember Induction Heuristic Algorithm (EIHA) is used to select a set of Strong Lattice Independent (SLI) vectors, which can be assumed to be Affine Independent, and therefore candidates to be the endmembers of the data. Selected endmembers are used to compute the linear unmixing of the robot's acquired images. The resulting mixing coefficients are used as feature vectors for view recognition through classification. We show on a sample path experiment that our approach can recognise the localization of the robot and we compare the results with the Independent Component Analysis (ICA).

  13. Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesions.

    PubMed Central

    Nerlich, A.; Schleicher, E.

    1991-01-01

    The immunohistochemical localization of the extracellular matrix was examined in 31 cases with different degrees of human diabetic nephropathy using antisera to human collagen types I, III, IV, V, fibronectin, laminin, and basement-membrane-associated heparan sulfate proteoglycan (HSPG). In normal glomeruli, HSPG was predominantly localized in the glomerular basement membrane and in the mesangium, and to minor extent in the basement membranes of tubules and Bowman's capsule. Collagen IV and laminin were distributed in glomerular basement membrane and mesangium in minor amounts. Interstitial collagens usually do not occur within glomeruli except for collagen V which has a light microscopic glomerular distribution similar to collagen IV. In diabetic diffuse glomerulosclerosis, the enlarged mesangial matrix showed an increased staining reaction for collagen IV, V, laminin, and fibronectin whereas the staining pattern of HSPG was markedly reduced. Early, small nodular lesions in diabetic glomeruli were similarly positive for most of the basement membrane components, whereas HSPG remained absent. With an increase in the diameter of the noduli, however, the staining reaction for all basement membrane components diminished, whereas interstitial collagens V and III, but not collagen I, were present in these noduli in substantial amounts. These initial studies provide evidence that the changes in the glomerular matrix in diabetic nephropathy may be divided into distinct and progressing stages of lesions. The reduced amount of HSPG even in slight, early lesions may represent the morphologic correlate to the impaired filter function of the glomerular basement membrane. Images Figure 3 Figure 5 Figure 1 Figure 2 Figure 4 PMID:1928305

  14. The interaction of local anesthetics with lipid membranes.

    PubMed

    Zapata-Morin, Patricio A; Sierra-Valdez, F J; Ruiz-Suárez, J C

    2014-09-01

    Molecular Dynamic Simulations are performed to evaluate the interaction of lidocaine, procaine and tetracaine with a lipid membrane. The main interest is to evaluate the structural changes produced by these local anesthetics in the bilayers. Penetration trajectories, interaction energies, entropy changes and an order parameter are calculated to quantify the destabilization of the lipid configurations. We show that such structural parameters give important information to understand how anesthetic agents influence the structure of plasma membranes. Graphic processing units (GPUs) are used in our simulations. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. A Phenotypic High-Content Screening Assay to Identify Regulators of Membrane Protein Localization.

    PubMed

    Smith, Lorey K; Thomas, Daniel W; Simpson, Kaylene J; Humbert, Patrick O

    2016-10-01

    Correct subcellular localization of proteins is a requirement for appropriate function. This is especially true in epithelial cells, which rely on the precise localization of a diverse array of epithelial polarity and cellular adhesion proteins. Loss of cell polarity and adhesion is a hallmark of cancer, and mislocalization of core polarity proteins, such as Scribble, is observed in a range of human epithelial tumors and is prognostic of poor survival. Despite this, little is known about how Scribble membrane localization is regulated. Here, we describe the development and application of a phenotypic high-content screening assay that is designed to specifically quantify membrane levels of Scribble to identify regulators of its membrane localization. A screening platform that is capable of resolving individual cells and quantifying membrane protein localization in confluent epithelial monolayers was developed by using the cytoplasm-to-cell-membrane bioapplication integrated with the Cellomics ArrayScan high-content imaging platform. Application of this method to a boutique human epithelial polarity and signaling small interfering RNA (siRNA) library resulted in highly robust coefficient-of-variance and Z' factor values. As proof of concept, we present two candidate genes whose depletion specifically reduces Scribble protein levels at the membrane. Data mining revealed that these proteins interact with components of the Scribble polarity complex, providing support for the utility of the screening approach. This method is broadly applicable to genome-wide and large-scale compound screening of membrane-bound proteins, and when coupled with pathway analysis the dataset becomes even more valuable and can provide predictive mechanistic insight.

  16. PRINCIPAL COMPONENTS FOR NON-LOCAL MEANS IMAGE DENOISING.

    PubMed

    Tasdizen, Tolga

    2008-01-01

    This paper presents an image denoising algorithm that uses principal component analysis (PCA) in conjunction with the non-local means image denoising. Image neighborhood vectors used in the non-local means algorithm are first projected onto a lower-dimensional subspace using PCA. Consequently, neighborhood similarity weights for denoising are computed using distances in this subspace rather than the full space. This modification to the non-local means algorithm results in improved accuracy and computational performance. We present an analysis of the proposed method's accuracy as a function of the dimensionality of the projection subspace and demonstrate that denoising accuracy peaks at a relatively low number of dimensions.

  17. Local measurements of phase transitions in Bacteriorhodopsin membrane

    NASA Astrophysics Data System (ADS)

    Proksch, R.; Nikiforov, M. P.; Hohlbauch, S.; King, W. P.; Antoraz Contera, S.; Voïtchovsky, K.; Kalinin, S. V.

    2010-03-01

    Phase transitions play an important role in biology. Specifically the thermodynamic stability of internal membrane proteins is an important issue of biophysics. Purple membrane from Halobacterium halobium contain bacteriorhodopsin (bR), an integral protein 70-80% of whole mass is intramembraneous. There are heated debates in the field about the parameters of thermal denaturation of bR, such as the denaturation temperature, enthalpy etc. Recently, bR was proposed as a component of biomolecular electronics. Thus, reliable information about the phase transitions of supported samples of bR membranes is necessary. Phase transitions in polymer/biopolymer materials are associated with the large changes in mechanical properties of the samples. We developed the technique for the measurements of the temperature dependence of the mechanical properties with high spatial resolution. This technique is based on the measurements of the contact stiffness of the atomic force microscopy tip -- sample system as a function of temperature.

  18. Local Spectral Component Decomposition for Multi-Channel Image Denoising.

    PubMed

    Rizkinia, Mia; Baba, Tatsuya; Shirai, Keiichiro; Okuda, Masahiro

    2016-07-01

    We propose a method for local spectral component decomposition based on the line feature of local distribution. Our aim is to reduce noise on multi-channel images by exploiting the linear correlation in the spectral domain of a local region. We first calculate a linear feature over the spectral components of an M -channel image, which we call the spectral line, and then, using the line, we decompose the image into three components: a single M -channel image and two gray-scale images. By virtue of the decomposition, the noise is concentrated on the two images, and thus our algorithm needs to denoise only the two gray-scale images, regardless of the number of the channels. As a result, image deterioration due to the imbalance of the spectral component correlation can be avoided. The experiment shows that our method improves image quality with less deterioration while preserving vivid contrast. Our method is especially effective for hyperspectral images. The experimental results demonstrate that our proposed method can compete with the other state-of-the-art denoising methods.

  19. Membrane localization and topology of the Yersinia pestis YscJ lipoprotein.

    PubMed

    Silva-Herzog, Eugenia; Ferracci, Franco; Jackson, Michael W; Joseph, Sabrina S; Plano, Gregory V

    2008-02-01

    The localization and membrane topology of the Yersinia pestis YscJ lipoprotein, an essential component of the type III secretion apparatus, was investigated. YscJ was demonstrated to be an inner membrane (IM) lipoprotein that is anchored to the periplasmic face of the IM via an N-terminal lipid moiety and via a C-terminal transmembrane (TM) domain. Localization of the N-terminal lipid moiety to the IM occurred regardless of the amino-acid residues found in the +2 or +3 positions. IM localization was dependent upon an intact N-terminal domain (amino acids +1 to +61), suggesting that this region plays a role in YscJ localization. In contrast, the YscJ C-terminal domain and TM domain were not required for IM localization. N-terminal sequence analysis demonstrated that a significant proportion of membrane-localized YscJ lacks N-acylation, the final modification required for Lol-dependent transport of a lipoprotein to the OM. Interestingly, attachment of the N-terminus to the IM was required for YscJ function; however, the YscJ secretion signal and lipo-box could be functionally replaced by the first TM domain of the YscV protein, suggesting that the mechanism of attachment to the IM was not critical.

  20. Lectins as membrane components of mitochondria from Ricinus communis.

    PubMed Central

    Bowles, D J; Schnarrenberger, C; Kauss, H

    1976-01-01

    1. Mitochondria were isolated from developing endosperm of Ricinus communis and were fractionated into outer membrane and inner membrane. The relative purity of the two membrane fractions was determined by marker enzymes. The fractions were also examined by negative-stain electron microscopy. 2. Membrane fractions were sequentially extracted in the following way. (a) Suspension in 0.5M-potassium phosphate, pH7.1; (b)suspension in 0.1M-EDTA (disodium salt)/0.05M-potassium phosphate, pH7.1; (c) sonication in 0.05M-potassium phosphate, pH7.1;(d)sonication in aq. Triton X-100 (0.1%). The membranes were pelleted by centrifugation at 100 000g for 15 min, between each step. Agglutination activity in the extracts was investigated by using trypsin-treated rabbit erythrocytes. 3. The addition of lactose to inner mitochondrial membrane resulted in the solubilization of part of the lectin activity, indicating that the protein was attached to the membrane via its carbohydrate-binding site. Pretreatment of the membranes with lactose before tha usual extraction procedure showed that lactose could extract lectins that normally required more harsh treatment of the membrane for solubilization. 4. Lectins extracted from inner membranes were purified by affinity chromatography on agarose gel. Polyacrylamide-gel electrophoresis of purified samples in sodium dodecyl sulphate indicated that at least part of the lectin present in inner mitochondrial membrane was identical with the R. communis agglutinin of mol.wt. 120 000. Images PLATE 1 PLATE 2 PMID:1008861

  1. Lectins as membrane components of mitochondria from Ricinus communis.

    PubMed

    Bowles, D J; Schnarrenberger, C; Kauss, H

    1976-11-15

    1. Mitochondria were isolated from developing endosperm of Ricinus communis and were fractionated into outer membrane and inner membrane. The relative purity of the two membrane fractions was determined by marker enzymes. The fractions were also examined by negative-stain electron microscopy. 2. Membrane fractions were sequentially extracted in the following way. (a) Suspension in 0.5M-potassium phosphate, pH7.1; (b)suspension in 0.1M-EDTA (disodium salt)/0.05M-potassium phosphate, pH7.1; (c) sonication in 0.05M-potassium phosphate, pH7.1;(d)sonication in aq. Triton X-100 (0.1%). The membranes were pelleted by centrifugation at 100 000g for 15 min, between each step. Agglutination activity in the extracts was investigated by using trypsin-treated rabbit erythrocytes. 3. The addition of lactose to inner mitochondrial membrane resulted in the solubilization of part of the lectin activity, indicating that the protein was attached to the membrane via its carbohydrate-binding site. Pretreatment of the membranes with lactose before tha usual extraction procedure showed that lactose could extract lectins that normally required more harsh treatment of the membrane for solubilization. 4. Lectins extracted from inner membranes were purified by affinity chromatography on agarose gel. Polyacrylamide-gel electrophoresis of purified samples in sodium dodecyl sulphate indicated that at least part of the lectin present in inner mitochondrial membrane was identical with the R. communis agglutinin of mol.wt. 120 000.

  2. Local and exotic components of primitive meteorites, and their origin

    NASA Technical Reports Server (NTRS)

    Anders, E.

    1987-01-01

    The chemical and isotopic compositions of chondrites and their implications for chondrite origins are considered, reviewing the results of recent theoretical and experimental investigations. Numerical data are compiled in tables and graphs and discussed in detail. Consideration is given to the properties and classifications of chondrites; components of local origin in the matrix, chondrules, Ca-Al-rich inclusions, and carbon and organic matter; exotic components such as O-16-enriched dust, nucleosynthetic anomalies, and extinct radionuclides; and isotopic anomalies of volatiles such as C, N, and the noble gases. The implications of the chondrite data for the stony fraction of comet nuclei are briefly indicated.

  3. Component local velocities of a ship along a defined axis

    NASA Astrophysics Data System (ADS)

    Crossland, P.; Lloyd, A. R. J. M.

    1991-02-01

    A method of calculating the component local velocities along a given axis when a ship is traveling at forward speed in long crested waves, is outlined. The results presented appear reasonable and illustrate expected trends. Only root mean square velocities along an axis are considered and not motions perpendicular to the defined axis. The theory and its subsequent inclusion into PAT-91 (ship motion computer program) are recorded in this technical memorandum.

  4. Amphiphilic components of the Murchison carbonaceous chondrite - Surface properties and membrane formation

    NASA Technical Reports Server (NTRS)

    Deamer, D. W.; Pashley, R. M.

    1989-01-01

    The possibility that the amphiphilic compounds in carbonaceous meteorites whose physicochemical properties are presently studied may represent sources of lipidlike compounds which could have evolved as membrane components in primitive cells is investigated in samples of the Murchison CM2 chondrite. Surface properties and membrane formation are obtained for three fractions isolated by two-dimensional thin-layer chromatography. It is concluded that a small, undefined fraction of Murchison components exhibits amphiphilic properties which allow assembly into boundary membranes.

  5. [Collagen type IV: major component of basement membranes. Current knowledge].

    PubMed

    Mercier, P; Ekindjian, O G

    1990-01-01

    The collagen family represents the most abundant protein in animals. Type IV collagen ([alpha 1 (IV)]2 alpha 2 (IV)) differs from the other types in several respects and particularly in its distribution, being strictly limited to the basement membranes. Alterations in the structure and functions of basement membranes are observed in a number of diseases, such as tumoral angiogenesis and diabetic nephropathy. This article reviews current structural and pathophysiological knowledge concerning type IV collagen.

  6. Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

    PubMed Central

    Chen, Allan L.; Kim, Elliot W.; Toh, Justin Y.; Vashisht, Ajay A.; Rashoff, Andrew Q.; Van, Christina; Huang, Amy S.; Moon, Andy S.; Bell, Hannah N.; Bentolila, Laurent A.; Wohlschlegel, James A.

    2015-01-01

    ABSTRACT The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. PMID:25691595

  7. SNAP-Tag-Reactive Lipid Anchors Enable Targeted and Spatiotemporally Controlled Localization of Proteins to Phospholipid Membranes.

    PubMed

    Rudd, Andrew K; Valls Cuevas, Joan M; Devaraj, Neal K

    2015-04-22

    The natural mechanisms that direct proteins to membranes are typically complex, requiring multiple steps and accessory components. It would be advantageous to develop simplified methods to direct proteins of interest to phospholipid membranes in a single step. Here we report a modular method for membrane localization of proteins by using chemically modified phospholipid anchors capable of covalent attachment to O(6)-methylguanine DNA methyltransferase (SNAP-tag) fusion proteins. To our knowledge, this is the first use of SNAP-tag reactions to modify benzylguanine-functionalized lipid membranes. We demonstrate that photocaged lipid precursors enable light-triggered spatial and temporal control over protein localization. The anchoring system is compatible with cell-free expression, allowing for genetic targeting of proteins to lipid membranes of giant unilamellar vesicles. This technique can be used to control membrane curvature effects, similar to what has been previously observed with certain membrane-bound proteins. This work addresses a current need in synthetic biology for simplified and robust methods to control membrane localization of expressed proteins and shows promise as a general tool for protein targeting to lipid vesicles and cellular membranes.

  8. A Local Learning Rule for Independent Component Analysis

    NASA Astrophysics Data System (ADS)

    Isomura, Takuya; Toyoizumi, Taro

    2016-06-01

    Humans can separately recognize independent sources when they sense their superposition. This decomposition is mathematically formulated as independent component analysis (ICA). While a few biologically plausible learning rules, so-called local learning rules, have been proposed to achieve ICA, their performance varies depending on the parameters characterizing the mixed signals. Here, we propose a new learning rule that is both easy to implement and reliable. Both mathematical and numerical analyses confirm that the proposed rule outperforms other local learning rules over a wide range of parameters. Notably, unlike other rules, the proposed rule can separate independent sources without any preprocessing, even if the number of sources is unknown. The successful performance of the proposed rule is then demonstrated using natural images and movies. We discuss the implications of this finding for our understanding of neuronal information processing and its promising applications to neuromorphic engineering.

  9. A Local Learning Rule for Independent Component Analysis

    PubMed Central

    Isomura, Takuya; Toyoizumi, Taro

    2016-01-01

    Humans can separately recognize independent sources when they sense their superposition. This decomposition is mathematically formulated as independent component analysis (ICA). While a few biologically plausible learning rules, so-called local learning rules, have been proposed to achieve ICA, their performance varies depending on the parameters characterizing the mixed signals. Here, we propose a new learning rule that is both easy to implement and reliable. Both mathematical and numerical analyses confirm that the proposed rule outperforms other local learning rules over a wide range of parameters. Notably, unlike other rules, the proposed rule can separate independent sources without any preprocessing, even if the number of sources is unknown. The successful performance of the proposed rule is then demonstrated using natural images and movies. We discuss the implications of this finding for our understanding of neuronal information processing and its promising applications to neuromorphic engineering. PMID:27323661

  10. Modulation of hematopoiesis by lymphocyte membrane-derived components.

    PubMed

    Guha, A; Mason, R P; Chen, L; Tuck, D P; Dainiak, N

    1994-04-01

    Membrane bound erythroid burst-promoting activity (mBPA) is an integral membrane protein that is present on normal B-cells and some activated T-cells, that induces burst-forming units-erythroid (BFU-E) when cultured with human erythropoietin (rHuEpo). Plasma membranes and vesicles shed from the leukemic A-1 cell line express mBPA. This activity derived from both A-1 cells and normal B-cells can be immunoadsorbed by the D3A4 antibody raised against mBPA. In this study, we demonstrate that interferon-gamma (IFN-gamma) suppresses BFU-E proliferation when added directly to culture of normal human bone marrow cells and in the absence and presence of A-1 cells. However, FACS analysis reveals that IFN-gamma enhances the surface expression of mBPA on A-1 cells. The role of IFN-gamma in modulating erythropoiesis in vitro is discussed with respect to the role of shedding membrane-derived vesicles from the B-cell surface.

  11. Intrarenal localization of the plasma membrane ATP channel pannexin1.

    PubMed

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T X; Yu, Alan; Peti-Peterdi, János

    2012-11-15

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1(-/-) mice. Urinary ATP levels were reduced by 30% in Panx1(-/-) compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions.

  12. Intrarenal localization of the plasma membrane ATP channel pannexin1

    PubMed Central

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T. X.; Yu, Alan

    2012-01-01

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1−/− mice. Urinary ATP levels were reduced by 30% in Panx1−/− compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions. PMID:22952282

  13. Plasma membrane-localized transporter for aluminum in rice

    PubMed Central

    Xia, Jixing; Yamaji, Naoki; Kasai, Tomonari; Ma, Jian Feng

    2010-01-01

    Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions, such as manganese, iron, and cadmium, or the Al–citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips except epidermal cells. Knockout of Nrat1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity, but did not affect the tolerance to other metals. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1). We therefore concluded that Nrat1 is a plasma membrane-localized transporter for trivalent Al, which is required for a prior step of final Al detoxification through sequestration of Al into vacuoles. PMID:20937890

  14. Plasma membrane-localized transporter for aluminum in rice.

    PubMed

    Xia, Jixing; Yamaji, Naoki; Kasai, Tomonari; Ma, Jian Feng

    2010-10-26

    Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions, such as manganese, iron, and cadmium, or the Al-citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips except epidermal cells. Knockout of Nrat1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity, but did not affect the tolerance to other metals. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1). We therefore concluded that Nrat1 is a plasma membrane-localized transporter for trivalent Al, which is required for a prior step of final Al detoxification through sequestration of Al into vacuoles.

  15. Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

    PubMed Central

    Taylor, M L; Duarte-Escalante, E; Reyes-Montes, M R; Elizondo, N; Maldonado, G; Zenteno, E

    1998-01-01

    The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris–HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal d-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68 kD and 180 kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for β-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues. PMID:9737672

  16. Novel components of the Toxoplasma inner membrane complex revealed by BioID.

    PubMed

    Chen, Allan L; Kim, Elliot W; Toh, Justin Y; Vashisht, Ajay A; Rashoff, Andrew Q; Van, Christina; Huang, Amy S; Moon, Andy S; Bell, Hannah N; Bentolila, Laurent A; Wohlschlegel, James A; Bradley, Peter J

    2015-02-17

    The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is

  17. Integrin clustering as a result of local membrane deformations and local signaling feedbacks

    NASA Astrophysics Data System (ADS)

    Felizzi, Federico; Iber, Dagmar

    2014-08-01

    Integrins are essential receptors for the development and functioning of multicellular animals because they mediate cell adhesion and migration, and regulate cell proliferation and apoptosis. Ligand-dependent activation of integrins involves the formation of receptor clusters and this has been accounted both to extracellular forces as mediated by the glycocalyx as well as to intracellular forces mediated by the cytoskeleton. Here we describe a Monte Carlo simulation that considers both the binding processes on the membrane as well as the intracellular signaling processes that stabilize the open integrin conformation. We show that integrin clustering can result both from the effects of integrin avidity, as a result of membrane deformations, as well as from the locally enhanced availability of talins in the open conformation, as a result of local positive feedback signaling via PIPKIγ and PIP2. The model was carefully parameterized based on reported quantitative data and reproduces a wide range of experimental data, including results that previously appeared inconsistent.

  18. Adaptive Decomposition of Highly Resolved Time Series into Local and Non‐local Components

    EPA Science Inventory

    Highly time-resolved air monitoring data are widely being collected over long time horizons in order to characterizeambient and near-source air quality trends. In many applications, it is desirable to split the time-resolved data into two ormore components (e.g., local and region...

  19. Adaptive Decomposition of Highly Resolved Time Series into Local and Non‐local Components

    EPA Science Inventory

    Highly time-resolved air monitoring data are widely being collected over long time horizons in order to characterizeambient and near-source air quality trends. In many applications, it is desirable to split the time-resolved data into two ormore components (e.g., local and region...

  20. Concentration Fluctuation in a Two-Component Fluid Membrane Surrounded with Three-Dimensional Fluids

    NASA Astrophysics Data System (ADS)

    Inaura, Keiichiro; Fujitani, Youhei

    2008-11-01

    We calculate the relaxation rate of the critical concentration fluctuation in a two-component fluid membrane by considering hydrodynamics of the surrounding fluids. Results are compared with the previous results obtained by Seki, Komura, and Imai (2007), who treated the momentum flux from the membrane to its environments using the friction coefficient.

  1. Dynamic Localization of Tat Protein Transport Machinery Components in Streptomyces coelicolor

    PubMed Central

    Willemse, Joost; Ruban-Ośmialowska, Beata; Widdick, David; Celler, Katherine; Hutchings, Matthew I.; van Wezel, Gilles P.

    2012-01-01

    The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle. PMID:23002216

  2. Locating Local Earthquakes Using Single 3-Component Broadband Seismological Data

    NASA Astrophysics Data System (ADS)

    Das, S. B.; Mitra, S.

    2015-12-01

    We devised a technique to locate local earthquakes using single 3-component broadband seismograph and analyze the factors governing the accuracy of our result. The need for devising such a technique arises in regions of sparse seismic network. In state-of-the-art location algorithms, a minimum of three station recordings are required for obtaining well resolved locations. However, the problem arises when an event is recorded by less than three stations. This may be because of the following reasons: (a) down time of stations in a sparse network; (b) geographically isolated regions with limited logistic support to setup large network; (c) regions of insufficient economy for financing multi-station network and (d) poor signal-to-noise ratio for smaller events at most stations, except the one in its closest vicinity. Our technique provides a workable solution to the above problematic scenarios. However, our methodology is strongly dependent on the velocity model of the region. Our method uses a three step processing: (a) ascertain the back-azimuth of the event from the P-wave particle motion recorded on the horizontal components; (b) estimate the hypocentral distance using the S-P time; and (c) ascertain the emergent angle from the vertical and radial components. Once this is obtained, one can ray-trace through the 1-D velocity model to estimate the hypocentral location. We test our method on synthetic data, which produces results with 99% precision. With observed data, the accuracy of our results are very encouraging. The precision of our results depend on the signal-to-noise ratio (SNR) and choice of the right band-pass filter to isolate the P-wave signal. We used our method on minor aftershocks (3 < mb < 4) of the 2011 Sikkim earthquake using data from the Sikkim Himalayan network. Location of these events highlight the transverse strike-slip structure within the Indian plate, which was observed from source mechanism study of the mainshock and larger aftershocks.

  3. Local anesthetics structure-dependently interact with anionic phospholipid membranes to modify the fluidity.

    PubMed

    Tsuchiya, Hironori; Ueno, Takahiro; Mizogami, Maki; Takakura, Ko

    2010-01-05

    While bupivacaine is more cardiotoxic than other local anesthetics, the mechanistic background for different toxic effects remains unclear. Several cardiotoxic compounds act on lipid bilayers to change the physicochemical properties of membranes. We comparatively studied the interaction of local anesthetics with lipid membranous systems which might be related to their structure-selective cardiotoxicity. Amide local anesthetics (10-300 microM) were reacted with unilamellar vesicles which were prepared with different phospholipids and cholesterol of varying lipid compositions. They were compared on the potencies to modify membrane fluidity by measuring fluorescence polarization. Local anesthetics interacted with liposomal membranes to increase the fluidity. Increasing anionic phospholipids in membranes enhanced the membrane-fluidizing effects of local anesthetics with the potency being cardiolipin>phosphatidic acid>phosphatidylglycerol>phosphatidylserine. Cardiolipin was most effective on bupivacaine, followed by ropivacaine. Local anesthetics interacted differently with biomimetic membranes consisting of 10mol% cardiolipin, 50mol% other phospholipids and 40mol% cholesterol with the potency being bupivacaine>ropivacaine>lidocaine>prilocaine, which agreed with the rank order of cardiotoxicity. Bupivacaine significantly fluidized 2.5-12.5mol% cardiolipin-containing membranes at cardiotoxicologically relevant concentrations. Bupivacaine is considered to affect lipid bilayers by interacting electrostatically with negatively charged cardiolipin head groups and hydrophobically with phospholipid acyl chains. The structure-dependent interaction with lipid membranes containing cardiolipin, which is preferentially localized in cardiomyocyte mitochondrial membranes, may be a mechanistic clue to explain the structure-selective cardiotoxicity of local anesthetics.

  4. Evaluation of stem cell components in retrocorneal membranes.

    PubMed

    Lee, Seok Hyun; Kim, Kyoung Woo; Kim, Mi Kyung; Chun, Yeoun Sook; Kim, Jae Chan

    2014-06-01

    The purpose of this study was to elucidate the origin and cellular composition of retrocorneal membranes (RCMs) associated with chemical burns using immunohistochemical staining for primitive cell markers. Six cases of RCMs were collected during penetrating keratoplasty. We examined RCMs with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) staining and immunohistochemical analysis using monoclonal antibodies against hematopoietic stem cells (CD34, CD133, c-kit), mesenchymal stem cells (beta-1-integrin, TGF-β, vimentin, hSTRO-1), fibroblasts (FGF-β, α-smooth muscle actin), and corneal endothelial cells (type IV collagen, CD133, VEGF, VEGFR1). Histologic analysis of RCMs revealed an organized assembly of spindle-shaped cells, pigment-laden cells, and thin collagenous matrix structures. RCMs were positive for markers of mesenchymal stem cells including beta-1-integrin, TGF-β, vimentin, and hSTRO-1. Fibroblast markers were also positive, including FGF-β and α-smooth muscle actin (SMA). In contrast, immunohistochemical staining was negative for hematopoietic stem cell markers including CD34, CD133 and c-kit as well as corneal endothelial cell markers such as type IV collagen, CD133 except VEGF and VEGFR1. Pigment-laden cells did not stain with any antibodies. The results of this study suggest that RCMs consist of a thin collagen matrix and fibroblast-like cells and may be a possible neogenetic structure produced from a lineage of bone marrow-derived mesenchymal stem cells.

  5. SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel

    PubMed Central

    Qiu, Zhaozhu; Dubin, Adrienne E.; Mathur, Jayanti; Tu, Buu; Reddy, Kritika; Miraglia, Loren J.; Reinhardt, Jürgen; Orth, Anthony P.; Patapoutian, Ardem

    2014-01-01

    Summary Maintenance of a constant cell volume in response to extracellular or intracellular osmotic changes is critical for cellular homeostasis. Activation of a ubiquitous volume-regulated anion channel (VRAC) plays a key role in this process; however, its molecular identity in vertebrates remains unknown. Here, we used a cell-based fluorescence assay and performed a genome-wide RNAi screen to find components of VRAC. We identified SWELL1 (LRRC8A), a member of a four-transmembrane protein family with unknown function, as essential for hypotonicity-induced iodide influx. SWELL1 is localized to the plasma membrane, and its knockdown dramatically reduces endogenous VRAC currents and regulatory cell volume decrease in various cell types. Furthermore, point mutations in SWELL1 cause a significant change in VRAC anion selectivity, demonstrating that SWELL1 is an essential VRAC component. These findings enable further molecular characterization of the VRAC channel complex and genetic studies for understanding the function of VRAC in normal physiology and disease. PMID:24725410

  6. Architecture, component, and microbiome of biofilm involved in the fouling of membrane bioreactors.

    PubMed

    Inaba, Tomohiro; Hori, Tomoyuki; Aizawa, Hidenobu; Ogata, Atsushi; Habe, Hiroshi

    2017-01-01

    Biofilm formation on the filtration membrane and the subsequent clogging of membrane pores (called biofouling) is one of the most persistent problems in membrane bioreactors for wastewater treatment and reclamation. Here, we investigated the structure and microbiome of fouling-related biofilms in the membrane bioreactor using non-destructive confocal reflection microscopy and high-throughput Illumina sequencing of 16S rRNA genes. Direct confocal reflection microscopy indicated that the thin biofilms were formed and maintained regardless of the increasing transmembrane pressure, which is a common indicator of membrane fouling, at low organic-loading rates. Their solid components were primarily extracellular polysaccharides and microbial cells. In contrast, high organic-loading rates resulted in a rapid increase in the transmembrane pressure and the development of the thick biofilms mainly composed of extracellular lipids. High-throughput sequencing revealed that the biofilm microbiomes, including major and minor microorganisms, substantially changed in response to the organic-loading rates and biofilm development. These results demonstrated for the first time that the architectures, chemical components, and microbiomes of the biofilms on fouled membranes were tightly associated with one another and differed considerably depending on the organic-loading conditions in the membrane bioreactor, emphasizing the significance of alternative indicators other than the transmembrane pressure for membrane biofouling.

  7. Isolation of hepatic mitochondrial contact sites: previously unrecognized inner membrane components.

    PubMed

    Hoppel, Charles; Kerner, Janos; Turkaly, Peter; Minkler, Paul; Tandler, Bernard

    2002-03-01

    An improved, fast, and relatively simple procedure for isolation of hepatic mitochondrial contact sites is described. These contact sites include conventional outer membrane, but the inner membrane component (which we term fusion patches) has a unique biochemical composition characterized by a clustering of three specific inner membrane proteins of 54, 52, and 31 kDa identified by proteomics, respectively, as the alpha and beta subunits of ATP synthase and the liver isoform of adenine nucleotide transferase. The contact site fraction was prepared using a discontinuous sucrose gradient from crude outer membranes derived from swollen/shrunk rat liver mitochondria. The resultant contact sites were analyzed using a continuous sucrose density gradient, revealing an apparent heterogeneity due to varying amounts of retained fusion patches in relation to the unvarying outer membrane component. By electron microscopy, contact sites consist of small vacuoles that contain one or several tiny vesicles, many of which are composed of multiple, closely packed lamellae. The contact site subfraction morphology is consistent with the biochemical variation. Thus, contact sites are not haphazard fusions of outer and inner membrane, but consist in part of regions of inner membrane of novel composition (fusion patches) and of conventional outer membrane.

  8. Local atomic structures of single-component metallic glasses

    NASA Astrophysics Data System (ADS)

    Trady, Salma; Hasnaoui, Abdellatif; Mazroui, M.'hammed; Saadouni, Khalid

    2016-10-01

    In this study we examine the structural properties of single-component metallic glasses of aluminum. We use a molecular dynamics simulation based on semi-empirical many-body potential, derived from the embedded atom method (EAM). The radial distribution function (RDF), common neighbors analysis method (CNA), coordination number analysis (CN) and Voronoi tessellation are used to characterize the metal's local structure during the heating and cooling (quenching). The simulation results reveal that the melting temperature depends on the heating rate. In addition, atomic visualization shows that the structure of aluminum after fast quenching is in a glassy state, confirmed quantitatively by the splitting of the second peak of the radial distribution function, and by the appearance of icosahedral clusters observed via CNA technique. On the other hand, the Wendt-Abraham parameters are calculated to determine the glass transition temperature (Tg), which depends strongly on the cooling rate; it increases while the cooling rate increases. On the basis of CN analysis and Voronoi tessellation, we demonstrate that the transition from the Al liquid to glassy state is mainly due to the formation of distorted and perfect icosahedral clusters.

  9. Alterations in progesterone receptor membrane component 2 (PGRMC2) in the endometrium of macaques afflicted with advanced endometriosis.

    PubMed

    Keator, Christopher S; Mah, Kuni; Slayden, Ov D

    2012-06-01

    The hormonally driven expression and cell-specific localization patterns of the progesterone receptor membrane components (PGRMC1 and PGRMC2) in the macaque endometrium during the menstrual cycle are unknown. Additionally, the expression and localization patterns of PGRMC1 and PGRMC2 in the secretory eutopic endometrium of primates afflicted with endometriosis are also unknown. Therefore, we used real-time PCR to quantify transcript expression levels of the PGRMCs in well-defined samples of endometrium collected from artificially cycled macaques during the menstrual cycle, and in the secretory phase endometrium of naturally cycling macaques afflicted with endometriosis. In situ hybridization and immunocytochemistry were used to localize PGRMC1 and PGRMC2 mRNA and protein, respectively. We compared the patterns of expression and localization of the PGRMCs with the expression and localization patterns of nuclear progesterone receptor (PGR). PGRMC1 and PGR were elevated during the proliferative phases of the cycle, and then declined to nearly undetectable levels during the late secretory phase of the cycle. Levels of PGRMC2 were lowest during the proliferative phases of the cycle and then increased markedly during the secretory phases. Strong staining for PGRMC2 was localized to the luminal and glandular epithelia during the secretory phases. When compared with artificially cycled disease-free animals, macaques with endometriosis exhibited no changes in the expression or localization patterns for PGR and PGRMC1 but exhibited strikingly reduced levels of PGRMC2 transcript and altered intracellular staining patterns for the PGRMC2 protein. Collectively, these results suggest that membrane-bound PGRMC2 may provide a pathway of action that could potentially mediate the non-genomic effects of progesterone on the glandular epithelia during the secretory phase of the cycle. Further, reduced levels of membrane-bound PGRMC2 may be associated with the progesterone insensitivity

  10. Reversible control of current across lipid membranes by local heating

    PubMed Central

    Urban, Patrick; Kirchner, Silke R.; Mühlbauer, Christian; Lohmüller, Theobald; Feldmann, Jochen

    2016-01-01

    Lipid membranes are almost impermeable for charged molecules and ions that can pass the membrane barrier only with the help of specialized transport proteins. Here, we report how temperature manipulation at the nanoscale can be employed to reversibly control the electrical resistance and the amount of current that flows through a bilayer membrane with pA resolution. For this experiment, heating is achieved by irradiating gold nanoparticles that are attached to the bilayer membrane with laser light at their plasmon resonance frequency. We found that controlling the temperature on the nanoscale renders it possible to reproducibly regulate the current across a phospholipid membrane and the membrane of living cells in absence of any ion channels. PMID:26940847

  11. Reversible control of current across lipid membranes by local heating

    NASA Astrophysics Data System (ADS)

    Urban, Patrick; Kirchner, Silke R.; Mühlbauer, Christian; Lohmüller, Theobald; Feldmann, Jochen

    2016-03-01

    Lipid membranes are almost impermeable for charged molecules and ions that can pass the membrane barrier only with the help of specialized transport proteins. Here, we report how temperature manipulation at the nanoscale can be employed to reversibly control the electrical resistance and the amount of current that flows through a bilayer membrane with pA resolution. For this experiment, heating is achieved by irradiating gold nanoparticles that are attached to the bilayer membrane with laser light at their plasmon resonance frequency. We found that controlling the temperature on the nanoscale renders it possible to reproducibly regulate the current across a phospholipid membrane and the membrane of living cells in absence of any ion channels.

  12. Reversible control of current across lipid membranes by local heating.

    PubMed

    Urban, Patrick; Kirchner, Silke R; Mühlbauer, Christian; Lohmüller, Theobald; Feldmann, Jochen

    2016-03-04

    Lipid membranes are almost impermeable for charged molecules and ions that can pass the membrane barrier only with the help of specialized transport proteins. Here, we report how temperature manipulation at the nanoscale can be employed to reversibly control the electrical resistance and the amount of current that flows through a bilayer membrane with pA resolution. For this experiment, heating is achieved by irradiating gold nanoparticles that are attached to the bilayer membrane with laser light at their plasmon resonance frequency. We found that controlling the temperature on the nanoscale renders it possible to reproducibly regulate the current across a phospholipid membrane and the membrane of living cells in absence of any ion channels.

  13. The Core Components of Organelle Biogenesis and Membrane Transport in the Hydrogenosomes of Trichomonas vaginalis

    PubMed Central

    Rada, Petr; Doležal, Pavel; Jedelský, Petr L.; Bursac, Dejan; Perry, Andrew J.; Šedinová, Miroslava; Smíšková, Kateřina; Novotný, Marian; Beltrán, Neritza Campo; Hrdý, Ivan; Lithgow, Trevor; Tachezy, Jan

    2011-01-01

    Trichomonas vaginalis is a parasitic protist of the Excavata group. It contains an anaerobic form of mitochondria called hydrogenosomes, which produce hydrogen and ATP; the majority of mitochondrial pathways and the organellar genome were lost during the mitochondrion-to-hydrogenosome transition. Consequently, all hydrogenosomal proteins are encoded in the nucleus and imported into the organelles. However, little is known about the membrane machineries required for biogenesis of the organelle and metabolite exchange. Using a combination of mass spectrometry, immunofluorescence microscopy, in vitro import assays and reverse genetics, we characterized the membrane proteins of the hydrogenosome. We identified components of the outer membrane (TOM) and inner membrane (TIM) protein translocases include multiple paralogs of the core Tom40-type porins and Tim17/22/23 channel proteins, respectively, and uniquely modified small Tim chaperones. The inner membrane proteins TvTim17/22/23-1 and Pam18 were shown to possess conserved information for targeting to mitochondrial inner membranes, but too divergent in sequence to support the growth of yeast strains lacking Tim17, Tim22, Tim23 or Pam18. Full complementation was seen only when the J-domain of hydrogenosomal Pam18 was fused with N-terminal region and transmembrane segment of the yeast homolog. Candidates for metabolite exchange across the outer membrane were identified including multiple isoforms of the β-barrel proteins, Hmp35 and Hmp36; inner membrane MCF-type metabolite carriers were limited to five homologs of the ATP/ADP carrier, Hmp31. Lastly, hydrogenosomes possess a pathway for the assembly of C-tail-anchored proteins into their outer membrane with several new tail-anchored proteins being identified. These results show that hydrogenosomes and mitochondria share common core membrane components required for protein import and metabolite exchange; however, they also reveal remarkable differences that reflect the

  14. Identification of the components of a glycolytic enzyme metabolon on the human red blood cell membrane.

    PubMed

    Puchulu-Campanella, Estela; Chu, Haiyan; Anstee, David J; Galan, Jacob A; Tao, W Andy; Low, Philip S

    2013-01-11

    Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as "label transfer" that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.

  15. Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane*

    PubMed Central

    Puchulu-Campanella, Estela; Chu, Haiyan; Anstee, David J.; Galan, Jacob A.; Tao, W. Andy; Low, Philip S.

    2013-01-01

    Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction. PMID:23150667

  16. ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking.

    PubMed

    Schröter, Saskia; Beckmann, Sabrina; Schmitt, Hans Dieter

    2016-09-01

    COPI-coated vesicles mediate retrograde membrane traffic from the cis-Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER-resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V-dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI•Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites. © 2016 The Authors.

  17. Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation.

    PubMed

    Dong, Shi-Jun; Yi, Chen-Feng; Li, Hao

    2015-12-01

    During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C18:1) but lower levels of hexadecanoic (C16:0) and palmitelaidic (C16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. The correlation between photosensitizers' membrane localization, membrane-residing targets, and photosensitization efficiency

    NASA Astrophysics Data System (ADS)

    Ytzhak, Shany; Bernstein, Shoshana; Loew, Leslie M.; Ehrenberg, Benjamin

    2009-06-01

    Various tetrapyrroles act as photosensitizers by efficiently generating singlet oxygen. Hydrophobic or amphiphilic photosensitizers are taken up by cells and are usually located in various cellular lipid membranes. Passive uptake by a membrane depends on biophysical properties of the membrane, such as its composition, temperature, phase, fluidity, electric potential etc., as well as on the external solution's properties. Although the intrinsic lifetime of singlet oxygen in the membrane phase is 10-30 μs, depending on lipid composition, it escapes much faster out of the membrane into the external or internal aqueous medium, where its lifetime is <3 μs. Any damage that singlet oxygen might inflict to membrane constituents, i.e. proteins or lipids, must thus occur while it is diffusing in the membrane. As a result, photosensitization efficiency depends, among others, on the location of the sensitizer in the membrane. Singlet oxygen can cause oxidative damage to two classes of targets in the membrane: lipids and proteins. Depolarization of the Nernst electric potential on cells' membranes was observed, but it is not clear whether lipid oxidation is a relevant factor leading to abolishing the resting potential of cells' membranes and to their death. We present a study of the effect of membrane lipid composition and the dissipation of the electric potential that is generated across the membrane. We find a clear correlation between the structure and unsaturation of lipids and the leakage of the membrane, which can be caused by their photosensitized oxidization. We demonstrate here that when liposomes are composed of mixtures similar to natural membranes, and photosensitization is being carried out under usual PDT conditions, photodamage to the lipids is not likely to cause enhanced permeability of ions through the membrane, which could be a mechanism that leads to cell death.

  19. Localized ridge defect augmentation using human pericardium membrane and demineralized bone matrix.

    PubMed

    Vidyadharan, Arun Kumar; Ravindran, Anjana

    2014-01-01

    Patient wanted to restore her lost teeth with implants in the lower left first molar and second premolar region. Cone beam computerized tomography (CBCT) revealed inadequate bone width and height around future implant sites. The extraction socket of second premolar area revealed inadequate socket healing with sparse bone fill after 4 months of extraction. To evaluate the clinical feasibility of using a collagen physical resorbable barrier made of human pericardium (HP) to augment localized alveolar ridge defects for the subsequent placement of dental implants. Ridge augmentation was done in the compromised area using Puros® demineralized bone matrix (DBM) Putty with chips and an HP allograft membrane. Horizontal (width) and vertical hard tissue measurements with CBCT were recorded on the day of ridge augmentation surgery, 4 month and 7 months follow-up. Intra oral periapical taken 1 year after implant installation showed minimal crestal bone loss. Bone volume achieved through guided bone regeneration was a gain of 4.8 mm horizontally (width) and 6.8 mm vertically in the deficient ridge within a period of 7 months following the procedure. The results suggested that HP Allograft membrane may be a suitable component for augmentation of localized alveolar ridge defects in conjunction with DBM with bone chips.

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

    PubMed Central

    Deng, Qiong; Wu, Yong; Zhang, Zeng; Wang, Yue; Li, Minghua

    2017-01-01

    The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR) localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens. PMID:28642789

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

    PubMed

    Deng, Qiong; Wu, Yong; Zhang, Zeng; Wang, Yue; Li, Minghua; Liang, Hui; Gui, Yaoting

    2017-01-01

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

  2. Synaptic membrane rafts: traffic lights for local neurotrophin signaling?

    PubMed

    Zonta, Barbara; Minichiello, Liliana

    2013-10-18

    Lipid rafts, cholesterol and lipid rich microdomains, are believed to play important roles as platforms for the partitioning of transmembrane and synaptic proteins involved in synaptic signaling, plasticity, and maintenance. There is increasing evidence of a physical interaction between post-synaptic densities and post-synaptic lipid rafts. Localization of proteins within lipid rafts is highly regulated, and therefore lipid rafts may function as traffic lights modulating and fine-tuning neuronal signaling. The tyrosine kinase neurotrophin receptors (Trk) and the low-affinity p75 neurotrophin receptor (p75(NTR)) are enriched in neuronal lipid rafts together with the intermediates of downstream signaling pathways, suggesting a possible role of rafts in neurotrophin signaling. Moreover, neurotrophins and their receptors are involved in the regulation of cholesterol metabolism. Cholesterol is an important component of lipid rafts and its depletion leads to gradual loss of synapses, underscoring the importance of lipid rafts for proper neuronal function. Here, we review and discuss the idea that translocation of neurotrophin receptors in synaptic rafts may account for the selectivity of their transduced signals.

  3. Effect of catalyst layer defects on local membrane degradation in polymer electrolyte fuel cells

    NASA Astrophysics Data System (ADS)

    Tavassoli, Arash; Lim, Chan; Kolodziej, Joanna; Lauritzen, Michael; Knights, Shanna; Wang, G. Gary; Kjeang, Erik

    2016-08-01

    Aiming at durability issues of fuel cells, this research is dedicated to a novel experimental approach in the analysis of local membrane degradation phenomena in polymer electrolyte fuel cells, shedding light on the potential effects of manufacturing imperfections on this process. With a comprehensive review on historical failure analysis data from field operated fuel cells, local sources of iron oxide contaminants, catalyst layer cracks, and catalyst layer delamination are considered as potential candidates for initiating or accelerating the local membrane degradation phenomena. Customized membrane electrode assemblies with artificial defects are designed, fabricated, and subjected to membrane accelerated stress tests followed by extensive post-mortem analysis. The results reveal a significant accelerating effect of iron oxide contamination on the global chemical degradation of the membrane, but dismiss local traces of iron oxide as a potential stressor for local membrane degradation. Anode and cathode catalyst layer cracks are observed to have negligible impact on the membrane degradation phenomena. Notably however, distinct evidence is found that anode catalyst layer delamination can accelerate local membrane thinning, while cathode delamination has no apparent effect. Moreover, a substantial mitigating effect for platinum residuals on the site of delamination is observed.

  4. Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane

    PubMed Central

    Cathcart, Kelly; Patel, Amit; Dies, Hannah; Rheinstädter, Maikel C.; Fradin, Cécile

    2015-01-01

    Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol’s condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content. PMID:26529029

  5. MEMBRANE FILTER PROCEDURE FOR ENUMERATING THE COMPONENT GENERA OF THE COLIFORM GROUP IN SEAWATER

    EPA Science Inventory

    A facile, quantitative, membrane filter procedure (mC) for defining the distribution of coliform populations in seawater according to the component genera was developed. The procedure, which utilizes a series of in situ substrate tests to obviate the picking of colonies for ident...

  6. MEMBRANE FILTER PROCEDURE FOR ENUMERATING THE COMPONENT GENERA OF THE COLIFORM GROUP IN SEAWATER

    EPA Science Inventory

    A facile, quantitative, membrane filter procedure (mC) for defining the distribution of coliform populations in seawater according to the component genera was developed. The procedure, which utilizes a series of in situ substrate tests to obviate the picking of colonies for ident...

  7. Interim report re: component parts for proton-exchange membrane fuel cells

    SciTech Connect

    George Marchetti

    1999-10-01

    The purpose of the first phase of the grant project is to design, develop and test a simplified fuel cell electrode structure for use in proton-exchange membrane fuel cells (''PEMFC''). By simplifying the structure of the electrode, mass production manufacturing efficiencies can be brought into play which will result in significant cost reductions for this fuel cell component. With a reduction in the cost of this key fuel cell component overall costs for PEMFC's can be brought within the commercialization target range of about US$100 per kilowatt for the fuel cell stack. Fuel cell electrodes are necessarily ''multi-layered'' composites. Multi-layers are required because of the several functions that the electrode must be able to perform in the working PEM fuel cell. The current generation of state-of-the-art porous fuel cell electrodes for PEMFC's is comprised of three primary layers. The first layer is the catalyst layer. Since hydrogen is the fuel used in this project and air is used as the oxidant, the catalyst must be capable of adsorbing hydrogen and oxygen from the air. While work is constantly on-going with respect to new hydrogen or oxygen catalysts, the best available catalyst at present for both of the reactant gases is platinum. To be effective, the catalyst (1) must be exposed to a constant flow of the respective reactant gas; (2) must be in intimate contact with the proton-exchange membrane; and (3) must be a finely divided catalyst and have a large specific surface area, especially on the oxidant side where the electrochemical reaction is slower by several orders of magnitude. The second layer is the substrate layer. The substrate layer provides structural support for the finely divided catalyst. It also functions as an electronic junction for conducting electricity produced by the electrochemical reaction from the catalyst layer to the bipolar plate of the fuel cell. In state-of-the-art PEMFC's, this layer is comprised of carbon particles (onto which

  8. Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components.

    PubMed

    Head, Brian P; Patel, Hemal H; Roth, David M; Murray, Fiona; Swaney, James S; Niesman, Ingrid R; Farquhar, Marilyn G; Insel, Paul A

    2006-09-08

    Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, beta1-adrenergic receptors (beta1-AR), beta2-AR, Galpha(s), and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (beta-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and alpha-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/mum of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Galpha(s) from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G(s)-AC in lipid rafts/caveolae.

  9. Partitioning behavior of aromatic components in jet fuel into diverse membrane-coated fibers.

    PubMed

    Baynes, Ronald E; Xia, Xin-Rui; Barlow, Beth M; Riviere, Jim E

    2007-11-01

    Jet fuel components are known to partition into skin and produce occupational irritant contact dermatitis (OICD) and potentially adverse systemic effects. The purpose of this study was to determine how jet fuel components partition (1) from solvent mixtures into diverse membrane-coated fibers (MCFs) and (2) from biological media into MCFs to predict tissue distribution. Three diverse MCFs, polydimethylsiloxane (PDMS, lipophilic), polyacrylate (PA, polarizable), and carbowax (CAR, polar), were selected to simulate the physicochemical properties of skin in vivo. Following an appropriate equilibrium time between the MCF and dosing solutions, the MCF was injected directly into a gas chromatograph/mass spectrometer (GC-MS) to quantify the amount that partitioned into the membrane. Three vehicles (water, 50% ethanol-water, and albumin-containing media solution) were studied for selected jet fuel components. The more hydrophobic the component, the greater was the partitioning into the membranes across all MCF types, especially from water. The presence of ethanol as a surrogate solvent resulted in significantly reduced partitioning into the MCFs with discernible differences across the three fibers based on their chemistries. The presence of a plasma substitute (media) also reduced partitioning into the MCF, with the CAR MCF system being better correlated to the predicted partitioning of aromatic components into skin. This study demonstrated that a single or multiple set of MCF fibers may be used as a surrogate for octanol/water systems and skin to assess partitioning behavior of nine aromatic components frequently formulated with jet fuels. These diverse inert fibers were able to assess solute partitioning from a blood substitute such as media into a membrane possessing physicochemical properties similar to human skin. This information may be incorporated into physiologically based pharmacokinetic (PBPK) models to provide a more accurate assessment of tissue dosimetry of

  10. Structure of the poly-C9 component of the complement membrane attack complex

    NASA Astrophysics Data System (ADS)

    Dudkina, Natalya V.; Spicer, Bradley A.; Reboul, Cyril F.; Conroy, Paul J.; Lukoyanova, Natalya; Elmlund, Hans; Law, Ruby H. P.; Ekkel, Susan M.; Kondos, Stephanie C.; Goode, Robert J. A.; Ramm, Georg; Whisstock, James C.; Saibil, Helen R.; Dunstone, Michelle A.

    2016-02-01

    The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

  11. Structure of the poly-C9 component of the complement membrane attack complex

    PubMed Central

    Dudkina, Natalya V.; Spicer, Bradley A.; Reboul, Cyril F.; Conroy, Paul J.; Lukoyanova, Natalya; Elmlund, Hans; Law, Ruby H. P.; Ekkel, Susan M.; Kondos, Stephanie C.; Goode, Robert J. A.; Ramm, Georg; Whisstock, James C.; Saibil, Helen R.; Dunstone, Michelle A.

    2016-01-01

    The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion. PMID:26841934

  12. Structure of the poly-C9 component of the complement membrane attack complex.

    PubMed

    Dudkina, Natalya V; Spicer, Bradley A; Reboul, Cyril F; Conroy, Paul J; Lukoyanova, Natalya; Elmlund, Hans; Law, Ruby H P; Ekkel, Susan M; Kondos, Stephanie C; Goode, Robert J A; Ramm, Georg; Whisstock, James C; Saibil, Helen R; Dunstone, Michelle A

    2016-02-04

    The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

  13. Electrostatic Localization of RNA to Protocell Membranes by Cationic Hydrophobic Peptides

    PubMed Central

    Kamat, Neha P; Tobé, Sylvia; Hill, Ian T; Szostak, Jack W

    2015-01-01

    Cooperative interactions between RNA and vesicle membranes on the prebiotic earth may have led to the emergence of primitive cells. The membrane surface offers a potential platform for the catalysis of reactions involving RNA, but this scenario relies upon the existence of a simple mechanism by which RNA could become associated with protocell membranes. Here, we show that electrostatic interactions provided by short, basic, amphipathic peptides can be harnessed to drive RNA binding to both zwitterionic phospholipid and anionic fatty acid membranes. We show that the association of cationic molecules with phospholipid vesicles can enhance the local positive charge on a membrane and attract RNA polynucleotides. This phenomenon can be reproduced with amphipathic peptides as short as three amino acids. Finally, we show that peptides can cross bilayer membranes to localize encapsulated RNA. This mechanism of polynucleotide confinement could have been important for primitive cellular evolution. PMID:26223820

  14. Exploring the local elastic properties of bilayer membranes using molecular dynamics simulations.

    PubMed

    Pieffet, Gilles; Botero, Alonso; Peters, Günther H; Forero-Shelton, Manu; Leidy, Chad

    2014-11-13

    Membrane mechanical elastic properties regulate a variety of cellular processes involving local membrane deformation, such as ion channel function and vesicle fusion. In this work, we used molecular dynamics simulations to estimate the local elastic properties of a membrane. For this, we calculated the energy needed to extract a DOPE lipid molecule, modified with a linker chain, from a POPC bilayer membrane using the umbrella sampling technique. Although the extraction energy entails several contributions related not only to elastic deformation but also to solvation, careful analysis of the potential of mean force (PMF) allowed us to dissect the elastic contribution. With this information, we calculated an effective linear spring constant of 44 ± 4 kJ·nm(-2)·mol(-1) for the DOPC membrane, in agreement with experimental estimates. The membrane deformation profile was determined independently during the stretching process in molecular detail, allowing us to fit this profile to a previously proposed continuum elastic model. Through this approach, we calculated an effective membrane spring constant of 42 kJ·nm(-2)·mol(-1), which is in good agreement with the PMF calculation. Furthermore, the solvation energy we derived from the data is shown to match the solvation energy estimated from critical micelle formation constants. This methodology can be used to determine how changes in lipid composition or the presence of membrane modifiers can affect the elastic properties of a membrane at a local level.

  15. Methodology for model updating of mechanical components with local nonlinearities

    NASA Astrophysics Data System (ADS)

    Kurt, Mehmet; Eriten, Melih; McFarland, D. Michael; Bergman, Lawrence A.; Vakakis, Alexander F.

    2015-11-01

    In this work, we propose a new nonlinear model updating strategy based on global/local nonlinear system identification of the dynamics. The main objective of this study is to construct and update reduced-order models (ROM) of a dynamical system based solely on measured data. The approach relies on analyzing transient system responses (local dynamics) in the frequency-energy domain, and based on these, constructing damped frequency-energy plots - FEPs (global dynamics) under the assumption of weak damping. The system parameters are characterized and updated by matching the backbone branches of the FEPs with reduced-order model FEPs using experimental or computational data. The main advantage of this method is that the system model is updated solely based on simulation and/or experimental results. It follows that the approach is purely data-driven. By matching the frequency-energy dependences of the dynamics of the physical dynamical system and its reduced order model, we are able to identify, update and reconstruct not only the global features of the dynamics in the frequency and energy ranges of interest, but also the local dynamics, i.e., individual time series for specific initial or excitation conditions. Hence, this work paves the way toward a nonlinear model updating methodology with broad applicability. The main features of the proposed methodology are demonstrated with a system of nonlinearly coupled beams excited by a concentrated transient force.

  16. Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin.

    PubMed

    Degendorfer, Georg; Chuang, Christine Y; Hammer, Astrid; Malle, Ernst; Davies, Michael J

    2015-12-01

    Basement membranes (BM) are specialized extracellular matrices underlying endothelial cells in the artery wall. Laminin, the most abundant BM glycoprotein, is a structural and biologically active component. Peroxynitrous acid (ONOOH), a potent oxidizing and nitrating agent, is formed in vivo at sites of inflammation from superoxide and nitric oxide radicals. Considerable data supports ONOOH formation in human atherosclerotic lesions, and an involvement of this oxidant in atherosclerosis development and lesion rupture. These effects may be mediated, at least in part, via extracellular matrix damage. In this study we demonstrate co-localization of 3-nitrotyrosine (a product of tyrosine damage by ONOOH) and laminin in human atherosclerotic lesions. ONOOH-induced damage to BM was characterized for isolated murine BM, and purified murine laminin-111. Exposure of laminin-111 to ONOOH resulted in dose-dependent loss of protein tyrosine and tryptophan residues, and formation of 3-nitrotyrosine, 6-nitrotryptophan and the cross-linked material di-tyrosine, as detected by amino acid analysis and Western blotting. These changes were accompanied by protein aggregation and fragmentation as detected by SDS-PAGE. Endothelial cell adhesion to isolated laminin-111 exposed to 10 μM or higher levels of ONOOH was significantly decreased (~25%) compared to untreated controls. These data indicate that laminin is oxidized by equimolar or greater concentrations of ONOOH, with this resulting in structural and functional changes. These modifications, and resulting compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the structure of atherosclerotic lesions, and an increased propensity to rupture. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Mannose incorporation and lectin recognition of pronase-sensitive components in Micrococcus lysodeikticus (M. luteus) membranes.

    PubMed

    Guerrero, A; Muñoz, E; Larraga, V

    1986-02-01

    Isolated cytoplasmic membranes from Micrococcus lysodeikticus were able to incorporate [14C]mannose from GDP-[14C]mannose. Labelled mannose remained in the membrane fraction after its repeated washing and lipid extraction. Sodium dodecyl sulfate gel electrophoresis in 12% acrylamide showed a set of bands with molecular weights ranging from 230 000 to 19 000 which stained for protein and carbohydrate, and incorporated [14C]mannose. Some of these bands reacted with different lectins (concanavalin A, wheat germ agglutinin and ricin). Furthermore, the mannose was incorporated via a glycosylation pathway similar to that followed in eukaryotic system as shown by the preliminary identification of a lipid intermediate transferring the sugar to proteins and by the differential sensitivity to bacitracin and tunicamycin. These complex membrane components were sensitive to digestion with pronase. All the results presented suggest their glycoprotein nature.

  18. Outer Membrane Components of the Tad (Tight Adherence) Secreton of Aggregatibacter actinomycetemcomitans▿ †

    PubMed Central

    Clock, Sarah A.; Planet, Paul J.; Perez, Brenda A.; Figurski, David H.

    2008-01-01

    Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins. PMID:18055598

  19. An essential novel component of the noncanonical mitochondrial outer membrane protein import system of trypanosomatids

    PubMed Central

    Pusnik, Mascha; Mani, Jan; Schmidt, Oliver; Niemann, Moritz; Oeljeklaus, Silke; Schnarwiler, Felix; Warscheid, Bettina; Lithgow, Trevor; Meisinger, Chris; Schneider, André

    2012-01-01

    The mitochondrial outer membrane protein Tom40 is the general entry gate for imported proteins in essentially all eukaryotes. Trypanosomatids lack Tom40, however, and use instead a protein termed the archaic translocase of the outer mitochondrial membrane (ATOM). Here we report the discovery of pATOM36, a novel essential component of the trypanosomal outer membrane protein import system that interacts with ATOM. pATOM36 is not related to known Tom proteins from other organisms and mediates the import of matrix proteins. However, there is a group of precursor proteins whose import is independent of pATOM36. Domain-swapping experiments indicate that the N-terminal presequence-containing domain of the substrate proteins at least in part determines the dependence on pATOM36. Secondary structure profiling suggests that pATOM36 is composed largely of α-helices and its assembly into the outer membrane is independent of the sorting and assembly machinery complex. Taken together, these results show that pATOM36 is a novel component associated with the ATOM complex that promotes the import of a subpopulation of proteins into the mitochondrial matrix. PMID:22787278

  20. Membrane-Based Characterization of a Gas Component — A Transient Sensor Theory

    PubMed Central

    Lazik, Detlef

    2014-01-01

    Based on a multi-gas solution-diffusion problem for a dense symmetrical membrane this paper presents a transient theory of a planar, membrane-based sensor cell for measuring gas from both initial conditions: dynamic and thermodynamic equilibrium. Using this theory, the ranges for which previously developed, simpler approaches are valid will be discussed; these approaches are of vital interest for membrane-based gas sensor applications. Finally, a new theoretical approach is introduced to identify varying gas components by arranging sensor cell pairs resulting in a concentration independent gas-specific critical time. Literature data for the N2, O2, Ar, CH4, CO2, H2 and C4H10 diffusion coefficients and solubilities for a polydimethylsiloxane membrane were used to simulate gas specific sensor responses. The results demonstrate the influence of (i) the operational mode; (ii) sensor geometry and (iii) gas matrices (air, Ar) on that critical time. Based on the developed theory the case-specific suitable membrane materials can be determined and both operation and design options for these sensors can be optimized for individual applications. The results of mixing experiments for different gases (O2, CO2) in a gas matrix of air confirmed the theoretical predictions. PMID:24608004

  1. Distribution of sialic acid between sialoglycoproteins and other membrane components of different erythrocyte phenotypes.

    PubMed

    Udoh, A E

    1991-01-01

    The sialic acid content of erythrocytes of three different AB0 blood groups have been studied. The sialic acid contents of erythrocyte membranes containing 300 mg protein were determined and compared. Groups 0 (Rhesus negative), AB (both Rhesus negative and positive), and B (Rhesus negative) blood differed significantly (p less than 0.05) in total sialic acid content and in the distribution of sialic acid between sialoglycoproteins and other membrane components. Membrane materials containing 300 mg total protein showed sialic acid contents of 52.73 +/- 2.2 mumol sialic acid for group 0 (Rhesus negative) 34.77 +/- 1.16 mumol for group AB (Rh negative), 32.88 +/- 1.52 mumol for AB (Rh positive) and 21.23 +/- 0.84 mumol for B (Rh negative). In group 0 (Rh. neg.) membranes 39.4 +/- 1.4% of the total sialic acid was associated with the sialoglycoproteins. The percentage of sialic acids associated with sialoglycoproteins in other erythrocyte membranes were 77.7 +/- 1.3% for group B, and 55.6 +/- 1.0% and 56.4 +/- 1.8% for group AB (Rh. negative) and (Rh. positive) respectively. The changes appear to be independent of the Rhesus grouping but dependent on the AB0 grouping since membranes of the two Rhesus types of group AB had identical total sialic acid and sialoglycoproteins sialic acids. The sialic acid densities in sialoglycoproteins also differed from one erythrocyte type to another. Group 0 (Rh. negative) membrane sialoglycoproteins had sialic acid density of 140.5 +/- 3.1 nmol/mg compared to 71.7 +/- 1.2 nmol/mg for group B and 128.1 +/- 2.2 and 124.5 +/- 4.0 nmol/mg for group AB Rhesus negative and Rhesus positive respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Identification and Quantitation of Plasma Membrane Components: A Biochemical Experiment for Lipid Investigations

    NASA Astrophysics Data System (ADS)

    Keys, Susan

    2000-11-01

    In this biochemistry exercise, students isolate and investigate lipid components of erythrocyte membranes. Erythrocytes are separated from sheep's blood by centrifugation. Lipids are extracted using chloroform-methanol (2:1 by volume). Individual lipid components (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and cholesterol) are separated and identified by thin-layer chromatography using chloroform-methanol-acetic acid-0.9% NaCl (75:45:3:1 by volume) as solvent. Nanomolar amounts of phospholipids are detected and quantified using a spectrophotometric phosphorus assay. Students learn to construct and use a standard concentration curve and test its accuracy.

  3. Transport of pure components in pervaporation through a microporous silica membrane.

    PubMed

    Bettens, Ben; Dekeyzer, Sofie; Van der Bruggen, Bart; Degrève, Jan; Vandecasteele, Carlo

    2005-03-24

    The pervaporation mechanism of pure components through a commercial microporous silica membrane was studied by performing experiments using water, methanol, ethanol, 2-propanol, and n-propanol in the 40-80 degrees C temperature range. Experimental fluxes were correlated to feed temperature and viscosity. It was found that the permeation mechanism obeys the adsorption-diffusion description, covering both the microscopic models based on configurational (micropore) diffusion and on activated surface diffusion. The contribution of convection was negligible. Size parameters for the permeating molecules such as molecular weight, kinetic diameter, and effective diameter, which are expected to have an influence on diffusion, did not correlate with the flux, thus strongly emphasizing the importance of sorption as the rate-determining step for transport in the pervaporation process. This was confirmed by correlating parameters reflecting polarity with flux: an exponential relation between the Hansen polarity (especially the hydrogen bonding component) and the flux was observed. A similar correlation was found between the dielectric constant and the flux. Furthermore, the flux increases in the same direction as the hydrophilicity of the pure components (log P). The effects of membrane surface tension and contact angles are less outspoken, but experiments performed on glass supported and silica supported membrane top layers suggest an important influence of the sublayers on the flux.

  4. Change of membrane potential is not a component of the photophobic transduction chain in Halobacterium halobium.

    PubMed Central

    Oesterhelt, D; Marwan, W

    1987-01-01

    Long (20 to 50 microns) and bipolarly flagellated cells of Halobacterium halobium were stimulated locally by a focused beam of light, and the photophobic response was analyzed. The results demonstrate that two flagellar bundles did not react in a coordinated fashion. The light-induced stop response of a flagellar bundle only occurred if the stimulus was applied within 5 microns of the polar region. This excluded membrane potential changes from being causally involved in photophobic signalling and indicated that there is a diffusible messenger in the signal transduction chain which is subjected to decay. In addition, the photoreceptor may be localized at the polar end of the cell. Images PMID:3611021

  5. Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries.

    PubMed

    Hoang, Hanh H; Nickerson, Nicholas N; Lee, Vincent T; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P; Lory, Stephen

    2011-01-01

    In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect

  6. HER2 signaling regulates HER2 localization and membrane retention

    PubMed Central

    Jeong, Jaekwang; Kim, Wonnam; Kim, Lark Kyun; VanHouten, Joshua; Wysolmerski, John J.

    2017-01-01

    ErbB2/HER2/Neu is a receptor tyrosine kinase that is overexpressed in 25–30% of human breast cancers, usually associated with amplification of the ERBB2 gene. HER2 has no recognized ligands and heterodimers between HER2 and EGFR (ErbB1/HER1) or HER2 and ErbB3/HER3 are important in breast cancer. Unlike other ErbB family members, HER2 is resistant to internalization and degradation, and remains at the cell surface to signal for prolonged periods after it is activated. Although the mechanisms underlying retention of HER2 at the cell surface are not fully understood, prior studies have shown that, in order to avoid internalization, HER2 must interact with the chaperone, HSP90, and the calcium pump, PMCA2, within specific plasma membrane domains that protrude from the cell surface. In this report, we demonstrate that HER2 signaling, itself, is important for the formation and maintenance of membrane protrusions, at least in part, by maintaining PMCA2 expression and preventing increased intracellular calcium concentrations. Partial genetic knockdown of HER2 expression or pharmacologic inhibition of HER2 signaling causes the depletion of membrane protrusions and disruption of the interactions between HER2 and HSP90. This is associated with the ubiquitination of HER2, its internalization with EGFR or HER3, and its degradation. These results suggest a model by which some threshold of HER2 signaling is required for the formation and/or maintenance of multi-protein signaling complexes that reinforce and prolong HER2/EGFR or HER2/HER3 signaling by inhibiting HER2 ubiquitination and internalization. PMID:28369073

  7. Localization and proliferation of lymphatic vessels in the tympanic membrane in normal state and regeneration.

    PubMed

    Miyashita, Takenori; Burford, James L; Hong, Young-Kwon; Gevorgyan, Haykanush; Lam, Lisa; Mori, Nozomu; Peti-Peterdi, Janos

    2013-10-25

    We clarified the localization of lymphatic vessels in the tympanic membrane and proliferation of lymphatic vessels during regeneration after perforation of the tympanic membrane by using whole-mount imaging of the tympanic membrane of Prox1 GFP mice. In the pars tensa, lymphatic vessel loops surrounded the malleus handle and annulus tympanicus. Apart from these locations, lymphatic vessel loops were not observed in the pars tensa in the normal tympanic membrane. Lymphatic vessel loops surrounding the malleus handle were connected to the lymphatic vessel loops in the pars flaccida and around the tensor tympani muscle. Many lymphatic vessel loops were detected in the pars flaccida. After perforation of the tympanic membrane, abundant lymphatic regeneration was observed in the pars tensa, and these regenerated lymphatic vessels extended from the lymphatic vessels surrounding the malleus at day 7. These results suggest that site-specific lymphatic vessels play an important role in the tympanic membrane. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Renal localization of the membrane attack complex in systemic lupus erythematosus nephritis.

    PubMed

    Biesecker, G; Katz, S; Koffler, D

    1981-12-01

    The membrane attack complex (MAC) of the complement system was localized in both glomeruli and peritubular regions of 22 kidneys manifesting systemic lupus erythematosus (SLE) nephritis. A similar distribution was observed for immune complex markers (IgG, Clq, and C3) and MAC in glomeruli, although the deposits of MAC were more discrete and showed lesser immunofluorescence staining intensity compared with immunoglobulins and complement components. In contrast, peritubular immune complexes were present in only 7 out of 22 kidneys, involved comparatively small clusters of tubules, exhibited weaker immunofluorescence staining than MAC, and failed to correlate with interstitial foci of inflammation. Granular or irregular, linear aggregates of the MAC were observed at the periphery of larger groups of tubules contiguous to areas of interstitial inflammation. Comparable amounts of IgG, Clq, C3, and MAC were present in blood vessel walls in areas of fibrinoid necrosis. These data suggest that the MAC is a direct mediator of tissue injury occurring in renal glomeruli, tubules, and blood vessels. The discordance between immune complexes and MAC localized in the peritubular region, but not in glomeruli or blood vessels, raises the possibility that both immune complexes and nonimmune agents, such as bacterial antigens, may activate the classical or alternative complement pathways and thereby play a role in the pathogenesis of tubulointerstitial lesions of SLE nephritis.

  9. Golgi-localized KIAA0725p regulates membrane trafficking from the Golgi apparatus to the plasma membrane in mammalian cells.

    PubMed

    Sato, Sei-ichi; Inoue, Hiroki; Kogure, Takeshi; Tagaya, Mitsuo; Tani, Katsuko

    2010-11-05

    Mammals have three members of the intracellular phospholipase A(1) protein family (phosphatidic acid preferring-phospholipase A(1), p125, and KIAA0725p). In this study, we showed that KIAA0725p is localized in the Golgi, and is rapidly cycled between the Golgi and cytosol. Catalytic activity is important for targeting of KIAA0725p to Golgi membranes. RNA interference experiments suggested that KIAA0725p contributes to efficient membrane trafficking from the Golgi apparatus to the plasma membrane, but is not involved in brefeldin A-induced Golgi-to-endoplasmic reticulum retrograde transport. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Indole Localization in Lipid Membranes Revealed by Molecular Simulation

    PubMed Central

    Norman, Kristen E.; Nymeyer, Hugh

    2006-01-01

    It is commonly known that the amino acid residue tryptophan and its side-chain analogs, e.g., indole, are strongly attracted to the interfacial region of lipid bilayers. Phenylalanine and its side-chain analogs, e.g., benzene, do not localize in the interface but are distributed throughout the lipid bilayer. We use molecular dynamics to investigate the details of indole and benzene localization and orientation within a POPC bilayer and the factors that lead to their different properties. We identify three sites in the bilayer at which indole is localized: 1), a site in the interface near the glycerol moiety; 2), a weakly bound site in the interface near the choline moiety; and 3), a weakly bound site in the center of the bilayer's hydrocarbon core. Benzene is localized in the same three positions, but the most stable position is the hydrocarbon core followed by the site near the glycerol moiety. Transfer of indole from water to the hydrocarbon core shows a classic hydrophobic effect. In contrast, interfacial binding is strongly enthalpy driven. We use several different sets of partial charges to investigate the factors that contribute to indole's and benzene's orientational and spatial distribution. Our simulations show that a number of electrostatic interactions appear to contribute to localization, including hydrogen bonding to the lipid carbonyl groups, cation-π interactions, interactions between the indole dipole and the lipid bilayer's strong interfacial electric field, and nonspecific electrostatic stabilization due to a mismatch in the variation of the nonpolar forces and local dielectric with position in the bilayer. PMID:16815896

  11. C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

    PubMed Central

    Gong, Jihong; Lai, Ying; Li, Xiaohong; Wang, Mengxian; Leitz, Jeremy; Hu, Yachong; Zhang, Yunxiang; Choi, Ucheor B.; Cipriano, Daniel; Pfuetzner, Richard A.; Südhof, Thomas C.; Yang, Xiaofei; Brunger, Axel T.

    2016-01-01

    In presynaptic nerve terminals, complexin regulates spontaneous “mini” neurotransmitter release and activates Ca2+-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca2+-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents. PMID:27821736

  12. The Nogo-B receptor promotes Ras plasma membrane localization and activation.

    PubMed

    Zhao, B; Hu, W; Kumar, S; Gonyo, P; Rana, U; Liu, Z; Wang, B; Duong, W Q; Yang, Z; Williams, C L; Miao, Q R

    2017-06-15

    The localization of prenylated Ras at the plasma membrane promotes activation of Ras by receptor tyrosine kinases and stimulates oncogenic signaling by mutant Ras. The Nogo-B receptor (NgBR) is a transmembrane receptor that contains a conserved hydrophobic pocket. Here, we demonstrate that the NgBR promotes the membrane accumulation of Ras by directly binding prenylated Ras at the plasma membrane. We show that NgBR knockdown diminishes the membrane localization of Ras in multiple cell types. NgBR overexpression in NIH-3T3 fibroblasts increases membrane-associated Ras, induces the transformed phenotype in vitro, and promotes the formation of fibrosarcoma in nude mice. NgBR knockdown in human breast cancer cells reduces Ras membrane localization, inhibits epidermal growth factor (EGF)-stimulated Ras signaling and diminishes tumorigenesis of xenografts in nude mice. Our data demonstrate that NgBR is a unique receptor that promotes accumulation of prenylated Ras at the plasma membrane and promotes EGF pathways.

  13. Two components of muscarine-sensitive membrane current in rat sympathetic neurones.

    PubMed Central

    Brown, D A; Selyanko, A A

    1985-01-01

    Membrane currents induced by muscarine (Imus) were recorded in voltage-clamped neurones in isolated rat superior cervical ganglia. Two components of Imus were regularly recorded: an inward current resulting from inhibition of the outward K+ current, IM; and an outward current attributable to the reduction of a steady inward current. The presence of these two components caused a 'cross-over' in the current-voltage curves at -50 +/- 3 mV in neurones impaled with KCl-filled micro-electrodes or at -63 +/- 4 mV in neurones impaled with K-acetate-filled electrodes. Both components of Imus were prevented by atropine. Both persisted in Krebs solution containing tetrodotoxin (1 microM), Cd2+ (200 microM) or 0 Ca2+. When IM was inhibited by external Ba2+ or internal Cs+ only the outward component of Imus could be detected. This component reversed at +3 +/- 2 mV in cells impaled with CsCl-filled electrodes or at -20 +/- 3 mV in cells impaled with Cs-acetate-filled electrodes. The reversal potentials agreed with those for the currents induced by gamma-aminobutyric acid (+4 +/- 2 mV and -16 +/- 3 mV with CsCl and Cs acetate electrodes respectively). Replacement of external NaCl with Na acetate (so reducing external Cl- concentration ( [Cl-]o) from 155 to 22 mM) shifted the reversal potential for Imus by +25 and +14.5 mV in two cells impaled with CsCl-filled electrodes. A tenfold reduction of external [Na+] (by glucosamine replacement) did not significantly alter the reversal potential for Imus in KCl or CsCl-impaled cells. Under conditions where IM is already inhibited, the residual outward component of Imus can lead to hyperpolarization and inhibition of neuronal activity in unclamped cells. We conclude that both inward and outward components of Imus result from direct activation of muscarinic receptors on the ganglion cells. The inward component results from IM inhibition. We suggest that the outward component results from inhibition of another, voltage-independent current IX

  14. An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes.

    PubMed

    Schmitt, Paulina; Rosa, Rafael D; Destoumieux-Garzón, Delphine

    2016-05-01

    Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due to their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

  15. Two-component coarse-grained molecular-dynamics model for the human erythrocyte membrane.

    PubMed

    Li, He; Lykotrafitis, George

    2012-01-04

    We present a two-component coarse-grained molecular-dynamics model for simulating the erythrocyte membrane. The proposed model possesses the key feature of combing the lipid bilayer and the erythrocyte cytoskeleton, thus showing both the fluidic behavior of the lipid bilayer and the elastic properties of the erythrocyte cytoskeleton. In this model, three types of coarse-grained particles are introduced to represent clusters of lipid molecules, actin junctions, and band-3 complexes, respectively. The proposed model facilitates simulations that span large length scales (approximately micrometers) and timescales (approximately milliseconds). By tuning the interaction potential parameters, we were able to control the diffusivity and bending rigidity of the membrane model. We studied the membrane under shearing and found that at a low shear strain rate, the developed shear stress was due mainly to the spectrin network, whereas the viscosity of the lipid bilayer contributed to the resulting shear stress at higher strain rates. In addition, we investigated the effects of a reduced spectrin network connectivity on the shear modulus of the membrane.

  16. Interaction of miltefosine with the lipid and protein components of the erythrocyte membrane.

    PubMed

    Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Hansen, Daiane; Alonso, Antonio

    2013-05-01

    Miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although its mechanism of action remains poorly understood. Electron paramagnetic resonance spectroscopy of a spin-labeled lipid and a thiol-specific spin label showed that MT causes an increase in the molecular dynamics of erythrocyte ghost membranes and detergent-resistant membranes (DRMs) prepared from erythrocyte ghosts. In the vesicles of lipid raft constituents, it was shown that 20 mol % sphingomyelin could be replaced by 20 mol % MT with no change in the molecular dynamics. The effect of MT in DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-90 µg/mL), our measurements in the blood plasma indicated a hemolytic level of 2%-5%. The experiments indicated that MT acts predominantly on the protein component of the membrane. MT aggregates may wrap around the hydrophobic polypeptide chains, forming micelle-like structures that stabilize protein conformations more exposed to the solvent. Proteins with higher hydrophobicity may induce the penetration of the hydrophilic groups of MT into the membrane and cause it to rupture.

  17. Biological and Biophysical Properties of the Tumor-localizing Component of Hematoporphyrin Derivative1

    PubMed Central

    Kessel, David; Cheng, May-Ling

    2015-01-01

    Reverse-phase chromatography, aqueous gel exclusion, and nonaqueous gel exclusion were assessed as procedures for preparative fractionation of the tumor-localizing product hematoporphyrin derivative. Porphyrin accumulation, fluorescence, and photodynamic cytotoxicity were monitored using the murine Sarcoma 180 tumor. Aqueous gel exclusion chromatography can provide a hematoporphyrin derivative fraction enriched in the tumor-localizing component. A further enrichment occurs when this procedure is carried out at 55°C, but nonlocalizing porphyrins could not be eliminated. While providing a better separation, reverse-phase chromatography cannot provide a tumor-localizing fraction free from contaminating protoporphyrin. However, this and other contaminants can be eliminated from the tumor-localizing fraction via nonaqueous gel exclusion chromatography. This latter separation provides two tumor-localizing products: (a) a fast-eluting fraction enriched in the major photosensitizing component(s); and (b) a more complex slowly eluting fraction enriched in fluorescence localizers. PMID:4005843

  18. Localization of Membrane Proteins in the Cyanobacterium Synechococcus sp. PCC7942 (Radial Asymmetry in the Photosynthetic Complexes).

    PubMed

    Sherman, D. M.; Troyan, T. A.; Sherman, L. A.

    1994-09-01

    Localization of membrane proteins in the cyanobacterium Synechococcus sp. PCC7942 was determined by transmission electron microscopy utilizing immunocytochemistry with cells prepared by freeze-substitution. This preparation procedure maintained cellular morphology and permitted detection of cellular antigens with high sensitivity and low background. Synechococcus sp. PCC7942 is a unicellular cyanobacterium with thylakoids organized in concentric layers toward the periphery of the cell. Cytochrome oxidase was localized almost entirely in the cytoplasmic membrane, whereas a carotenoprotein (P35) was shown to be a cell wall component. The major photosystem II (PSII) proteins (D1, D2 CP43, and CP47) were localized throughout the thylakoids. Proteins of the Cyt b6/f complex were found to have a similar distribution. Thylakoid luminal proteins, such as the Mn-stabilizing protein, were located primarily in the thylakoid, but a small, reproducible fraction was found in the outer compartment. The photosystem I (PSI) reaction center proteins and the ATP synthase proteins were found associated mostly with the outermost thylakoid and with the cytoplasmic membrane. These results indicated that the photosynthetic apparatus is not evenly distributed throughout the thylakoids. Rather, there is a radial asymmetry such that much of the PSI and the ATPase synthase is located in the outermost thylakoid. The relationship of this structure to the photosynthetic mechanism is discussed. It is suggested that the photosystems are separated because of kinetic differences between PSII and PSI, as hypothesized by H.-W. Trissl and C. Wilhelm (Trends Biochem Sci [1993] 18:415-419).

  19. Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues.

    PubMed

    Coïc, Yves-Marie; Vincent, Michel; Gallay, Jacques; Baleux, Françoise; Mousson, Florence; Beswick, Veronica; Neumann, Jean-Michel; de Foresta, Béatrice

    2005-12-01

    Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We

  20. Intracellular transport of viruses and their components: utilizing the cytoskeleton and membrane highways.

    PubMed

    Harries, Phillip A; Schoelz, James E; Nelson, Richard S

    2010-11-01

    Plant viruses are obligate organisms that require host components for movement within and between cells. A mechanistic understanding of virus movement will allow the identification of new methods to control virus systemic spread and serve as a model system for understanding host macromolecule intra- and intercellular transport. Recent studies have moved beyond the identification of virus proteins involved in virus movement and their effect on plasmodesmal size exclusion limits to the analysis of their interactions with host components to allow movement within and between cells. It is clear that individual virus proteins and replication complexes associate with and, in some cases, traffic along the host cytoskeleton and membranes. Here, we review these recent findings, highlighting the diverse associations observed between these components and their trafficking capacity. Plant viruses operate individually, sometimes within virus species, to utilize unique interactions between their proteins or complexes and individual host cytoskeletal or membrane elements over time or space for their movement. However, there is not sufficient information for any plant virus to create a complete model of its intracellular movement; thus, more research is needed to achieve that goal.

  1. Solid-state nanopore localization by controlled breakdown of selectively thinned membranes

    NASA Astrophysics Data System (ADS)

    Carlsen, Autumn T.; Briggs, Kyle; Hall, Adam R.; Tabard-Cossa, Vincent

    2017-02-01

    We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid-state membranes by spatially varying the electric field strength with localized membrane thinning. We show 100 × 100 nm2 precision in standard SiN x membranes (30-100 nm thick) after selective thinning by as little as 25% with a helium ion beam. Control over nanopore position is achieved through the strong dependence of the electric field-driven CBD mechanism on membrane thickness. Confinement of pore formation to the thinned region of the membrane is confirmed by TEM imaging and by analysis of DNA translocations. These results enhance the functionality of CBD as a fabrication approach and enable the production of advanced nanopore devices for single-molecule sensing applications.

  2. PKCδ localization at the membrane increases matrix traction force dependent on PLCγ1/EGFR signaling.

    PubMed

    Jamison, Joshua; Lauffenburger, Douglas; Wang, James C-H; Wells, Alan

    2013-01-01

    During wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKCδ/MLC-2). PKCδ is activated by growth factor-driven PLCγ1 hydrolysis of phosphoinositide bisphosphate (PIP2) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKCδ n-terminus mediates PKCδ localization to the membrane in relative proximity to PLCγ1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKCδ mediates the transcellular contractility of fibroblasts. To determine PKCδ activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKCδ constructs were generated; PKCδ-CaaX with farnesylation moiety targeting PKCδ to the membrane and PKCδ-SaaX a non-targeting control. Increased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKCδ (PKCδ-CaaX) when analyzed with 2D cell traction force and 3D compaction of collagen matrix. This effect was reduced in cells deficient in EGFR/PLCy1 signaling. In cells expressing non-membrane targeted PKCδ (PKCδ-SaaX), the cell force exerted outside the ECM (extracellular matrix) was less, but cell motility/speed/persistence was increased after EGF stimulation. Change in cell motility and increased force exertion was also preceded by change in cell morphology. Organization of actin stress fibers was also decreased as a result of increasing membrane targeting of PKCδ. From these results membrane tethering of PKCδ leads to increased force exertion on ECM. Furthermore, our data show PLCγ1 regulation of PKCδ, at least in part, drives transcellular contractility in fibroblasts.

  3. Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane

    PubMed Central

    Lei, Jennifer; Priddy, Lauren B.; Lim, Jeremy J.; Massee, Michelle; Koob, Thomas J.

    2017-01-01

    Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION® Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek® activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair. PMID:28224047

  4. Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane.

    PubMed

    Lei, Jennifer; Priddy, Lauren B; Lim, Jeremy J; Massee, Michelle; Koob, Thomas J

    2017-02-01

    Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION(®) Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek(®) activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair.

  5. Local area water removal analysis of a proton exchange membrane fuel cell under gas purge conditions.

    PubMed

    Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

    2012-01-01

    In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold.

  6. Local Area Water Removal Analysis of a Proton Exchange Membrane Fuel Cell under Gas Purge Conditions

    PubMed Central

    Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

    2012-01-01

    In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold. PMID:22368495

  7. Structure of two-component lipid membranes on solid support: An x-ray reflectivity study

    SciTech Connect

    Novakova, Eva; Giewekemeyer, Klaus; Salditt, Tim

    2006-11-15

    We report an x-ray reflectivity study of phospholipid membranes deposited on silicon by vesicle fusion. The samples investigated were composed of single phospholipid bilayers as well as two-component lipid bilayer systems with varied charge density. We show that the resolution obtained in the density profile across the bilayer is high enough to distinguish two head-group maxima in the profile if the sample is in the phase coexistence regime. The water layer between the bilayer and silicon is found to depend on the lipid surface charge density.

  8. Scaling of membrane-type locally resonant acoustic metamaterial arrays.

    PubMed

    Naify, Christina J; Chang, Chia-Ming; McKnight, Geoffrey; Nutt, Steven R

    2012-10-01

    Metamaterials have emerged as promising solutions for manipulation of sound waves in a variety of applications. Locally resonant acoustic materials (LRAM) decrease sound transmission by 500% over acoustic mass law predictions at peak transmission loss (TL) frequencies with minimal added mass, making them appealing for weight-critical applications such as aerospace structures. In this study, potential issues associated with scale-up of the structure are addressed. TL of single-celled and multi-celled LRAM was measured using an impedance tube setup with systematic variation in geometric parameters to understand the effects of each parameter on acoustic response. Finite element analysis was performed to predict TL as a function of frequency for structures with varying complexity, including stacked structures and multi-celled arrays. Dynamic response of the array structures under discrete frequency excitation was investigated using laser vibrometry to verify negative dynamic mass behavior.

  9. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    PubMed

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

  10. ADP-ribosylation of membrane components by pertussis and cholera toxin

    SciTech Connect

    Ribeiro-Neto, F.A.P.; Mattera, F.; Hildebrandt, J.D.; Codina, J.; Field, J.B.; Birnbaumer, L.; Sekura, R.D.

    1985-01-01

    Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (N/sub s/) and inhibitory (N/sub i/) regulatory components of adenylyl cyclase. Cholera toxin acts on N/sub s/ by ADP-ribosylating its ..cap alpha../sub s/ subunit; pertussis toxin acts on N/sub i/ by ADP-ribosylating its ..cap alpha..; subunit. By using (/sup 32/P)NAD/sup +/ and determining the transfer of its (/sup 32/P)ADP-ribose moiety to membrane components, it is possible to obtain information on N/sub s/ and N/sub i/. A set of protocols is presented that can be used to study simultaneously and comparatively the susceptibility of N/sub s/ and N/sub i/ to be ADP-ribosylated by cholera and pertussis toxin.

  11. Two-component membrane material properties and domain formation from dissipative particle dynamics.

    PubMed

    Illya, G; Lipowsky, R; Shillcock, J C

    2006-09-21

    The material parameters (area stretch modulus and bending rigidity) of two-component amphiphilic membranes are determined from dissipative particle dynamics simulations. The preferred area per molecule for each species is varied so as to produce homogeneous mixtures or nonhomogeneous mixtures that form domains. If the latter mixtures are composed of amphiphiles with the same tail length, but different preferred areas per molecule, their material parameters increase monotonically as a function of composition. By contrast, mixtures of amphiphiles that differ in both tail length and preferred area per molecule form both homogeneous and nonhomogeneous mixtures that both exhibit smaller values of their material properties compared to the corresponding pure systems. When the same nonhomogeneous mixtures of amphiphiles are assembled into planar membrane patches and vesicles, the resulting domain shapes are different when the bending rigidities of the domains are sufficiently different. Additionally, both bilayer and monolayer domains are observed in vesicles. We conclude that the evolution of the domain shapes is influenced by the high curvature of the vesicles in the simulation, a result that may be relevant for biological vesicle membranes.

  12. Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes.

    PubMed

    Breukink, Eefjan; van Heusden, Hester E; Vollmerhaus, Pauline J; Swiezewska, Ewa; Brunner, Livia; Walker, Suzanne; Heck, Albert J R; de Kruijff, Ben

    2003-05-30

    The peptidoglycan layers surrounding bacterial membranes are essential for bacterial cell survival and provide an important target for antibiotics. Many antibiotics have mechanisms of action that involve binding to Lipid II, the prenyl chain-linked donor of the peptidoglycan building blocks. One of these antibiotics, the pore-forming peptide nisin uses Lipid II as a receptor molecule to increase its antimicrobial efficacy dramatically. Nisin is the first example of a targeted membrane-permeabilizing peptide antibiotic. However, it was not known whether Lipid II functions only as a receptor to recruit nisin to bacterial membranes, thus increasing its specificity for bacterial cells, or whether it also plays a role in pore formation. We have developed a new method to produce large amounts of Lipid II and variants thereof so that we can address the role of the lipid-linked disaccharide in the activity of nisin. We show here that Lipid II is not only the receptor for nisin but an intrinsic component of the pore formed by nisin, and we present a new model for the pore complex that includes Lipid II.

  13. Co-existence of Gel and Fluid Lipid Domains in Single-component Phospholipid Membranes

    SciTech Connect

    Armstrong, Clare L; Barrett, M; Toppozini, L; Yamani, Zahra; Kucerka, Norbert; Katsaras, John; Fragneto, Giovanna; Rheinstadter, Maikel C

    2012-01-01

    Lateral nanostructures in membranes, so-called rafts, are believed to strongly influence membrane properties and functions. The experimental observation of rafts has proven difficult as they are thought to be dynamic structures that likely fluctuate on nano- to microsecond time scales. Using neutron diffraction we present direct experimental evidence for the co-existence of gel and fluid lipid domains in a single-component phospholipid membrane made of DPPC as it undergoes its main phase transition. The coherence length of the neutron beam sets a lower limit for the size of structures that can be observed. Neutron coherence lengths between 30 and 242A used in this study were obtained by varying the incident neutron energy and the resolution of the neutron spectrometer. We observe Bragg peaks corresponding to co-existing nanometer sized structures, both in out-of-plane and in-plane scans, by tuning the neutron coherence length. During the main phase transition, instead of a continuous transition that shows a pseudo-critical behavior, we observe the co-existence of gel and fluid domains.

  14. Determination of the leaching of polymeric ion-selective membrane components by stripping voltammetry.

    PubMed

    Paczosa-Bator, Beata; Piech, Robert; Lewenstam, Andrzej

    2010-05-15

    This paper focuses on the quantitative determination of the loss of the components from plastic membranes of ion-selective electrodes (ISEs) during contact with aqueous bathing solutions. The leaching processes, which affect the ISE responses, are rarely characterized by independent methods. For this purpose, differential pulse cathodic stripping voltammetry (DP CSV) is used. This method, owing to its high sensitivity, acceptable recovery and accuracy, is a good tool to characterize the kinetics of leakage of the lipophilic salts. Sodium tetraphenylborate (NaTPB) leakage from the PVC-based sodium-selective membrane containing two different plasticizers, o-nitrophenyl octyl ether (o-NPOE) or di(2-ethylhexyl) sebacate (DOS) is presented. Correlation between the rate of leaching of the lipophilic salt and dielectric constants of the plasticizers is observed. The data obtained by DP CSV correlate well with potentiometric and electrochemical impedance responses. The observed outflow of TPB(-) is associated with decreasing potentiometric sensitivity to sodium and increasing bulk membrane resistance.

  15. The alpha2beta1 isoform of guanylyl cyclase mediates plasma membrane localized nitric oxide signalling.

    PubMed

    Bellingham, Michelle; Evans, Thomas J

    2007-10-01

    Nitric oxide (NO) is a mediator of copious biological processes, in many cases through the production of cGMP from the enzyme nitric oxide-sensitive guanylyl cyclase. Natriuretic peptides also elevate cGMP, often with distinct biological effects, raising the issue of how specificity is achieved. Here we show that a recently described alpha(2)beta(1) isoform of guanylyl cyclase is expressed in a number of epithelia, where it is localized to the apical plasma membrane. We measured the functional properties of the alpha(2)beta(1) isoform by utilizing the NO-dependent activation of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR), which occurs by phosphorylation via the membrane-bound type II isoform of cGMP-dependent protein kinase. We found that cGMP generated by NO activation of the alpha(2)beta(1) isoform of guanylyl cyclase is an exceptionally efficient mediator of nitric oxide action on membrane targets, activating CFTR far more effectively than the cytoplasmically located alpha(1)beta(1) guanylyl cyclase isoform. Targeting the alpha(1)beta(1) isoform of guanylyl cyclase to the membrane also dramatically enhanced the effects of nitric oxide on CFTR within the membrane. This was not due to increased enzymatic activity of guanylyl cyclase in a membrane location, but to production of a localised membrane pool of cGMP by membrane-localized NO-dependent guanylyl cyclase that was resistant to degradation by phosphodiesterases. Selective effects of cGMP produced from this enzyme in response to NO are directed at membrane targets and suggest that drugs selectively activating or inhibiting this alpha(2)beta(1) isoform of guanylyl cyclase may have unique pharmacological properties.

  16. Influence of local anesthetics on molecular organization in phosphatidylethanolamine membranes

    SciTech Connect

    Kelusky, E.C.; Smith, I.C.

    1984-09-01

    The influence of the local anesthetics tetracaine (TTC) and procaine (PRC) on bilayers of specifically deuterated phosphatidylethanolamines (PE) has been studied by /sup 2/H and /sup 31/P NMR. Dimyristoylphosphatidylethanolamines (DMPE), deuterated at positions 2, 4, and 14 of the sn-2 chain, position 2 of the sn-1 chain, and in the ethanolamine headgroup, were mixed 1:1 with a semisynthetic egg PE and the effect of measured quantities of TTC and PRC on the /sup 2/H quadrupole splittings, spin-lattice relaxation times, and /sup 31/P chemical shift anisotropy were observed. Experiments were performed at pH 5.5, when the anesthetics are primarily charged, and at pH 9.5, when they are uncharged. Tetracaine was observed to disorder the hydrocarbon region of the bilayer and to induce a conformational change in the PE headgroup. Conversely, procaine had little or no effect on the hydrocarbon region and induced only a small change in the headgroup. These conformational changes and disordering effects, when adjusted for anesthetic partitioning, are essentially independent of the charge on the anesthetic. However, at pH 5.5 and low TTC/PE molar ratios (less than 0.1), the /sup 2/H NMR spectra showed two lipid environments--one corresponding to free PE and the other to PE in contact with TTC. Continued addition of TTC resulted in the eventual disappearance of the free PE signal and the corresponding growth of the signal from PE in contact with TTC. At pH 9.5, when TTC is uncharged, only one signal is observed. In mixtures of PE and phosphatidylserine, a conformational change in the headgroup was noted which was similar to that seen in the pure PE; however, there was no evidence for slow lateral diffusion of the anesthetics.

  17. Chitosan facilitates structure formation of the salivary gland by regulating the basement membrane components.

    PubMed

    Yang, Tsung-Lin; Hsiao, Ya-Chuan

    2015-10-01

    Tissue structure is important for inherent physiological function and should be recapitulated during tissue engineering for regenerative purposes. The salivary gland is a branched organ that is responsible for saliva secretion and regulation. The salivary glands develop from epithelial-mesenchymal interactions, and depend on the support of the basement membrane (BM). Chitosan-based biomaterials have been demonstrated to be competent in facilitating the formation of salivary gland tissue structure. However, the underlying mechanisms have remained elusive. In the developing submandibular gland (SMG), the chitosan effect was found to diminish when collagen and laminin were removed from cultured SMG explants. Chitosan increased the expression of BM components including collagen, laminin, and heparan sulfate proteoglycan, and also facilitated BM components and the corresponding receptors to be expressed in tissue-specific patterns beneficial for SMG branching. The chitosan effect decreased when either laminin components or receptors were inhibited, as well when the downstream signaling was blocked. Our results revealed that chitosan promotes salivary glands branching through the BM. By regulating BM components and receptors, chitosan efficiently stimulated downstream signaling to facilitate salivary gland branching. The present study revealed the underlying mechanism of the chitosan effect in engineering SMG structure formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. [Iontophoresis - local anesthesia at the ear canal and tympanic membrane (author's transl)].

    PubMed

    Tolsdorff, P

    1980-02-01

    Operations to the external ear canal and tympanic membrane necessitate sufficient local anesthesia. General sedation followed by infiltration anestesia, is rather time-consumming, can be painful due to the injection, and is not particularly satisfactory for the treatment of outpatients. The iontophorese-technique, however, of local anesthesia, is applicable particularly for the treatment of outpatients. Principally, the local anesthetic is transported in ionisised form to the nerve membrane, by means of calvanic currents through the healthy surface epithelial tissue of the external ear canal or the eardrum. The technique described for the first time in 1911 no longer shows toxic sides-effects since the introduction of improved electrodes and more modern local anesthetic. The anatomic, pharmacological, chemical and physical basics of the technique will be described. The lecture will be based on personal experience of the method, taken from large groups of patients over a period of more than two years, using equipment specially designed for this purpose.

  19. Local-Level Prognostics Health Management Systems Framework for Passive AdvSMR Components. Interim Report

    SciTech Connect

    Ramuhalli, Pradeep; Roy, Surajit; Hirt, Evelyn H.; Pardini, Allan F.; Jones, Anthony M.; Deibler, John E.

    2014-09-12

    This report describes research results to date in support of the integration and demonstration of diagnostics technologies for prototypical AdvSMR passive components (to establish condition indices for monitoring) with model-based prognostics methods. The focus of the PHM methodology and algorithm development in this study is at the localized scale. Multiple localized measurements of material condition (using advanced nondestructive measurement methods), along with available measurements of the stressor environment, enhance the performance of localized diagnostics and prognostics of passive AdvSMR components and systems.

  20. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein.

    PubMed Central

    Berman, C L; Yeo, E L; Wencel-Drake, J D; Furie, B C; Ginsberg, M H; Furie, B

    1986-01-01

    We have identified and purified a platelet integral membrane protein (140,000 mol wt), using the KC4 monoclonal antibody specific for activated platelets, that is internal in resting platelets but exposed on activated platelets (Hsu-Lin S.-C., C.L. Berman, B.C. Furie, D. August, and B. Furie, 1984, J. Biol. Chem. 259: 9121-9126.). The expression of the protein on the platelet surface is secretion-dependent. This protein has been named platelet activation-dependent granule-external membrane (PADGEM) protein. PADGEM protein is distinct from the surface glycoproteins of resting platelets, but identical to the S12 antigen, GMP-140. Using immunofluorescent staining, resting platelets failed to stain for PADGEM protein with the KC4 antibody, but after permeabilization showed a punctate staining of the cell interior. Thrombin-stimulated intact platelets stained with a peripheral rim pattern thus demonstrating the translocation of PADGEM protein from an internal location to the cell surface. PADGEM protein expression on the platelet surface at varying thrombin concentrations correlated with alpha granule release, as measured by the secretion of platelet factor 4. Further evidence for an alpha granule localization of PADGEM protein was provided by nitrogen cavitation of resting platelets followed by metrizamide density gradient centrifugation; PADGEM protein codistributed with platelet factor 4. Using immunoelectron microscopy, the protein was localized to the alpha granule in frozen ultrathin sections of resting platelets labeled using rabbit anti-PADGEM protein antibodies, whereas in thrombin-activated platelets, the plasma membrane was labeled. These studies indicate that PADGEM protein is a component of the alpha granule membrane of resting platelets and is incorporated into the plasma membrane upon activation and secretion. Images PMID:2941452

  1. Local defects in the nanostructure of the membrane of erythrocytes upon ionizing radiation of blood

    NASA Astrophysics Data System (ADS)

    Kozlova, E. K.; Sergunova, V. A.; Krasavin, E. A.; Boreyko, A. V.; Zavialova, A. V.; Kozlov, A. P.; Chernysh, A. M.

    2016-01-01

    The purpose of the study is to investigate local topological defects in the erythrocyte membranes resulting from the ultraviolet (UV) radiation of blood in vitro. Biological effects in the erythrocytes after exposure to UV radiation at a wavelength of 254 nm are equivalent to those after γ radiation. It has been shown that oxidative processes developing in a suspension upon UV radiation result in the disruption of the nanostructure of the membranes of erythrocytes. In the experiments, typical topological defects in the membrane nanostructure were observed. The parameters of the defects differed from the characteristics of the nanostructure of the control cell membrane without irradiation. The characteristic dimensions of the topological defects are commensurate with the size of the spectrin matrix. As a result of the exposure to the UV radiation, polymorphism of the erythrocytes was observed.

  2. Cholesterol increase in mitochondria: its effect on inner-membrane functions, submitochondrial localization and ultrastructural morphology.

    PubMed Central

    Echegoyen, S; Oliva, E B; Sepulveda, J; Díaz-Zagoya, J C; Espinosa-García, M T; Pardo, J P; Martínez, F

    1993-01-01

    The effect of cholesterol incorporation on some functions of the mitochondrial inner membrane and on the morphology of rat liver mitochondria was studied. Basal ATPase and succinate dehydrogenase activities remained unchanged after cholesterol was incorporated into the mitochondria; however, uncoupled ATPase activity was partially inhibited. The presence of several substrates and inhibitors did not change the amount of cholesterol incorporated, which was localized mostly in the outer membrane. Electron-microscope observations revealed the presence of vesicles between the outer and inner membranes; these vesicles increased in number with the amount of cholesterol incorporated. The data suggest that cholesterol induces the formation of vesicles from the outer membrane, and modifies the activity of stimulated ATPase. Images Figure 4 PMID:8435069

  3. Phospholipase A2 from sheep erythrocyte membranes. Ca2+ dependence and localization.

    PubMed

    Frei, E; Zahler, P

    1979-02-02

    The calcium dependence and the time course of phosphatidylethanolamine and phosphatidylcholine degradation by sheep erythrocyte membrane suspensions in presence of Triton X-100 were investigated. One enzyme with phospholipase A2 specificity was found to be responsible for both phosphatidyl-ethanolamine and phosphatidylcholine degradation. The localization of this enzyme in the membrane of the sheep erythrocyte was investigated by proteolytic treatment of sealed erythrocyte ghosts from the outside and of ghosts which had both sides of the membrane exposed to chymotrypsin. The inability of sealed ghosts to take up chymotrypsin was followed by flux measurements of [14C]dextran carboxyl previously trapped in the ghosts. No efflux of the marker was found during the proteolytic treatment. By comparing the residual phospholipase activities in the membranes from both ghost preparations, we concluded that the phospholipase is oriented to the exterior of the sheep erythrocyte.

  4. Biochemical and cytochemical localization of ATPases on the membranes of the electrocyte of Electrophorus electricus.

    PubMed

    Somló, C; de Souza, W; Machado, R D; Hassón-Voloch, A

    1977-11-30

    The localization of (Na+-K+) ATPase in the intact electrocyte of the electric organ of Electrophorus electricus (L.) and its subcellular fractions was investigated by biochemical and cytochemical methods. The distribution of AChE activity in the subcellular fractions was also comparatively analysed with this enzyme serving as a marker of the innervated membranes of the electrocyte. After application of cytochemical method of Farquhar and Palade to glutaraldehyde-fixed tissue, reaction was observed only at the membranes of vesicles localized at the periphery of the electrocyte. Previously fixed electrocytes, incubated in Ernst's medium showed reaction only at the vesicles whereas in unfixed tissue reaction also appeared at other membranes (surface and invaginations) of the anterior and posterior faces. This reaction was significantly inhibited in the presence of ouabain or in the absence of K+. Inhibition of Na+-K+-ATPase by glutaraldehyde fixation was also confirmed by biochemical analysis.

  5. Expression and membrane localization of MCT isoforms along the length of the human intestine.

    PubMed

    Gill, Ravinder K; Saksena, Seema; Alrefai, Waddah A; Sarwar, Zaheer; Goldstein, Jay L; Carroll, Robert E; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

    2005-10-01

    Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of approximately 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells.

  6. Outer Membrane Targeting of Pseudomonas aeruginosa Proteins Shows Variable Dependence on the Components of Bam and Lol Machineries

    PubMed Central

    Hoang, Hanh H.; Nickerson, Nicholas N.; Lee, Vincent T.; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P.; Lory, Stephen

    2011-01-01

    ABSTRACT In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. PMID:22147293

  7. The MICOS component Mic60 displays a conserved membrane-bending activity that is necessary for normal cristae morphology

    PubMed Central

    Tarasenko, Daryna; Kroppen, Benjamin; Heim, Gudrun

    2017-01-01

    The inner membrane (IM) of mitochondria displays an intricate, highly folded architecture and can be divided into two domains: the inner boundary membrane adjacent to the outer membrane and invaginations toward the matrix, called cristae. Both domains are connected by narrow, tubular membrane segments called cristae junctions (CJs). The formation and maintenance of CJs is of vital importance for the organization of the mitochondrial IM and for mitochondrial and cellular physiology. The multisubunit mitochondrial contact site and cristae organizing system (MICOS) was found to be a major factor in CJ formation. In this study, we show that the MICOS core component Mic60 actively bends membranes and, when inserted into prokaryotic membranes, induces the formation of cristae-like plasma membrane invaginations. The intermembrane space domain of Mic60 has a lipid-binding capacity and induces membrane curvature even in the absence of the transmembrane helix. Mic60 homologues from α-proteobacteria display the same membrane deforming activity and are able to partially overcome the deletion of Mic60 in eukaryotic cells. Our results show that membrane bending by Mic60 is an ancient mechanism, important for cristae formation, and had already evolved before α-proteobacteria developed into mitochondria. PMID:28254827

  8. The MICOS component Mic60 displays a conserved membrane-bending activity that is necessary for normal cristae morphology.

    PubMed

    Tarasenko, Daryna; Barbot, Mariam; Jans, Daniel C; Kroppen, Benjamin; Sadowski, Boguslawa; Heim, Gudrun; Möbius, Wiebke; Jakobs, Stefan; Meinecke, Michael

    2017-04-03

    The inner membrane (IM) of mitochondria displays an intricate, highly folded architecture and can be divided into two domains: the inner boundary membrane adjacent to the outer membrane and invaginations toward the matrix, called cristae. Both domains are connected by narrow, tubular membrane segments called cristae junctions (CJs). The formation and maintenance of CJs is of vital importance for the organization of the mitochondrial IM and for mitochondrial and cellular physiology. The multisubunit mitochondrial contact site and cristae organizing system (MICOS) was found to be a major factor in CJ formation. In this study, we show that the MICOS core component Mic60 actively bends membranes and, when inserted into prokaryotic membranes, induces the formation of cristae-like plasma membrane invaginations. The intermembrane space domain of Mic60 has a lipid-binding capacity and induces membrane curvature even in the absence of the transmembrane helix. Mic60 homologues from α-proteobacteria display the same membrane deforming activity and are able to partially overcome the deletion of Mic60 in eukaryotic cells. Our results show that membrane bending by Mic60 is an ancient mechanism, important for cristae formation, and had already evolved before α-proteobacteria developed into mitochondria. © 2017 Tarasenko et al.

  9. Performance of Water Recirculation Loop Maintenance Components for the Advanced Spacesuit Water Membrane Evaporator

    NASA Technical Reports Server (NTRS)

    Rector, Tony; Peyton, Barbara M.; Steele, John W.; Makinen, Janice; Bue, Grant C.; Campbell, Colin

    2014-01-01

    Water loop maintenance components to maintain the water quality of the Advanced Spacesuit Water Membrane Evaporation (SWME) water recirculation loop have undergone a comparative performance evaluation with a recirculating control loop which had no water quality maintenance. Results show that periodic water maintenance can improve performance of the SWME. The SWME is a heat rejection device under development at the NASA Johnson Space Center to perform thermal control for advanced spacesuits. One advantage of this technology is the potential for a significantly greater degree of tolerance to contamination when compared to the existing sublimator technology. The driver for the evaluation of water recirculation maintenance components was to enhance the robustness of the SWME through the leveraging of fluid loop management lessons learned from the International Space Station (ISS). A patented bed design that was developed for a United Technologies Aerospace System military application provided a low pressure drop means for water maintenance in the SWME recirculation loop. The bed design is coupled with high capacity ion exchange resins, organic adsorbents, and a cyclic methodology developed for the Extravehicular Mobility Unit (EMU) Transport Water loop. The maintenance cycle included the use of a biocide delivery component developed for the ISS to introduce a biocide in a microgravity compatible manner for the Internal Active Thermal Control System (IATCS). The leveraging of these water maintenance technologies to the SWME recirculation loop is a unique demonstration of applying the valuable lessons learned on the ISS to the next generation of manned spaceflight Environmental Control and Life Support System (ECLSS) hardware.

  10. Experimental dissection of oxygen transport resistance in the components of a polymer electrolyte membrane fuel cell

    NASA Astrophysics Data System (ADS)

    Oh, Hwanyeong; Lee, Yoo il; Lee, Guesang; Min, Kyoungdoug; Yi, Jung S.

    2017-03-01

    Oxygen transport resistance is a major obstacle for obtaining high performance in a polymer electrolyte membrane fuel cell (PEMFC). To distinguish the major components that inhibit oxygen transport, an experimental method is established to dissect the oxygen transport resistance of the components of the PEMFC, such as the substrate, micro-porous layer (MPL), catalyst layer, and ionomer film. The Knudsen numbers are calculated to determine the types of diffusion mechanisms at each layer by measuring the pore sizes with either mercury porosimetry or BET analysis. At the under-saturated condition where condensation is mostly absent, the molecular diffusion resistance is dissected by changing the type of inert gas, and ionomer film permeation is separated by varying the inlet gas humidity. Moreover, the presence of the MPL and the variability of the substrate thickness allow the oxygen transport resistance at each component of a PEMFC to be dissected. At a low relative humidity of 50% and lower, an ionomer film had the largest resistance, while the contribution of the MPL was largest for the other humidification conditions.

  11. Dual effect of local anesthetics on the function of excitable rod outer segment disk membrane

    SciTech Connect

    Mashimo, T.; Abe, K.; Yoshiya, I.

    1986-04-01

    The effects of local anesthetics and a divalent cation, Ca2+, on the function of rhodopsin were estimated from the measurements of light-induced proton uptake. The light-induced proton uptake by rhodopsin in the rod outer segment disk membrane was enhanced at lower pH (4) but depressed at higher pHs (6 to 8) by the tertiary amine local anesthetics lidocaine, bupivacaine, tetracaine, and dibucaine. The order of local anesthetic-induced depression of the proton uptake followed that of their clinical anesthetic potencies. The depression of the proton uptake versus the concentration of the uncharged form of local anesthetic nearly describes the same curve for small and large dose of added anesthetic. Furthermore, a neutral local anesthetic, benzocaine, depressed the proton uptake at all pHs between 4 and 7. These results indicate that the depression of the proton uptake is due to the effect of only the uncharged form. It is hypothesized that the uncharged form of local anesthetics interacts hydrophobically with the rhodopsin in the disk membrane. The dual effect of local anesthetics on the proton uptake, on the other hand, suggests that the activation of the function of rhodopsin may be caused by the charged form. There was no significant change in the light-induced proton uptake by rhodopsin when 1 mM of Ca2+ was introduced into the disk membrane at varying pHs in the absence or presence of local anesthetics. This fact indicates that Ca2+ ion does not influence the diprotonating process of metarhodopsin; neither does it interfere with the local anesthetic-induced changes in the rhodopsin molecule.

  12. Crystal Structure of Escherichia coli CusC the Outer Membrane Component of a Heavy Metal Efflux Pump

    SciTech Connect

    R Kulathila; R Kulathila; M Indic; B van den Berg

    2011-12-31

    While copper has essential functions as an enzymatic co-factor, excess copper ions are toxic for cells, necessitating mechanisms for regulating its levels. The cusCBFA operon of E. coli encodes a four-component efflux pump dedicated to the extrusion of Cu(I) and Ag(I) ions. We have solved the X-ray crystal structure of CusC, the outer membrane component of the Cus heavy metal efflux pump, to 2.3 {angstrom} resolution. The structure has the largest extracellular opening of any outer membrane factor (OMF) protein and suggests, for the first time, the presence of a tri-acylated N-terminal lipid anchor. The CusC protein does not have any obvious features that would make it specific for metal ions, suggesting that the narrow substrate specificity of the pump is provided by other components of the pump, most likely by the inner membrane component CusA.

  13. Modeling the local potential at Pt nanoparticles in polymer electrolyte membranes.

    PubMed

    Eslamibidgoli, Mohammad Javad; Melchy, Pierre-Éric Alix; Eikerling, Michael H

    2015-04-21

    We present a physical-analytical model for the potential distribution at Pt nanodeposits in a polymer electrolyte membrane (PEM). Experimental studies have shown that solid deposits of Pt in PEM play a dual role in radical-initiated membrane degradation. Surface reactions at Pt particles could facilitate the formation as well as the scavenging of ionomer-attacking radical species. The net radical balance depends on local equilibrium conditions at Pt nanodeposits in the PEM, specifically, their equivalent local electrode potential. Our approach utilizes a continuum description of crossover fluxes of reactant gases, coupled with the kinetics of electrochemical surface reactions at Pt nanodeposits to calculate the potential distribution. The local potential is a function of the PEM structure and composition, which is determined by PEM thickness, concentrations of H2 and O2, as well as the size and density distribution of Pt particles. Model results compare well with experimental data for the potential distribution in PEMs.

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

    PubMed Central

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

    2015-01-01

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

  15. Hydrodynamic Effect on Concentration Fluctuation in a Two-Component Fluid Membrane with a Spherical Shape

    NASA Astrophysics Data System (ADS)

    Fujitani, Youhei

    2013-01-01

    To the Gaussian model of a two-component fluid membrane with a spherical shape, we apply the mode coupling theory to study the hydrodynamic effect on the relaxation coefficient of concentration fluctuation in the homogeneous phase close to the critical point. In particular, when the viscosities of the three-dimensional fluids are the same inside and outside the vesicle, we obtain a concise analytical expression representing the hydrodynamic effect on the smallest wave-number mode. We derive its approximate expressions in various parameter regions to discuss the size effect of the vesicle. Much larger wave-number modes are studied numerically by means of our theoretical result. It is suggested that the hydrodynamic effect be diffusion-like, irrespective of the vesicle size, as long as the wavelength is much longer than the correlation length.

  16. Germ-line specific variants of components of the mitochondrial outer membrane import machinery in Drosophila.

    PubMed

    Hwa, Jennifer J; Zhu, Alan J; Hiller, Mark A; Kon, Charlene Y; Fuller, Margaret T; Santel, Ansgar

    2004-08-13

    A search of the Drosophila genome for genes encoding components of the mitochondrial translocase of outer membrane (TOM) complex revealed duplication of genes encoding homologues of Tom20 and Tom40. Tom20 and Tom40 were represented by two differentially expressed homologues in the Drosophila genome. While dtom20 and dtom40 appeared to be expressed ubiquitously, the second variants, called tomboy20 and tomboy40, were expressed only in the male germ-line. Transcripts for tomboy20 and tomboy40 were detected in primary spermatocytes as well as post-meiotic stages. Transcription of tomboy20 and tomboy40 in spermatocytes was not dependent on the transcription factor Cannonball, which is responsible for controlling expression of gene products exclusively required for post-meiotic germ cell differentiation. Epitope-tagging and transient expression of dTom20 and Tomboy40 in mammalian cell culture showed proper targeting to mitochondria.

  17. Ionic strength dependence of localized contact formation between membranes: nonlinear theory and experiment.

    PubMed Central

    Coakley, W T; Gallez, D; de Souza, E R; Gauci, H

    1999-01-01

    Erythrocyte membrane surface or suspending phase properties can be experimentally modified to give either spatially periodic local contacts or continuous contact along the seams of interacting membranes. Here, for cells suspended in a solution of the uncharged polysaccharide dextran, the average lateral separation between localized contacts in spatially periodic seams at eight ionic strengths, decreasing from 0.15 to 0.065, increased from 0.65 to 3.4 micrometers. The interacting membranes and intermembrane aqueous layer were modeled as a fluid film, submitted to a disjoining pressure, responding to a displacement perturbation either through wave growth resulting in spatially periodic contacts or in perturbation decay, to give a plane continuous film. Measured changes of lateral contact separations with ionic strength change were quantitatively consistent with analytical predictions of linear theory for an instability mechanism dependent on the membrane bending modulus. Introduction of a nonlinear approach established the consequences of the changing interaction potential experienced by different parts of the membrane as the disturbance grew. Numerical solutions of the full nonlinear governing equations correctly identified the ionic strength at which the bifurcation from continuous seam to a stationary periodic contact pattern occurred and showed a decrease in lateral contact and wave crest separation with increasing ionic strength. The nonlinear approach has the potential to recognize the role of nonspecific interactions in initiating the localized approach of membranes, and then incorporate the contribution of specific molecular interactions, of too short a range to influence the beginning of perturbation growth. This new approach can be applied to other biological processes such as neural cell adhesion, phagocytosis, and the acrosome reaction. PMID:10423428

  18. Local elasticity and adhesion of nanostructures on Drosophila melanogaster wing membrane studied using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Wagner, Ryan; Pittendrigh, Barry R.; Raman, Arvind

    2012-10-01

    Insect wings have a naturally occurring, complex, functional, hierarchical microstructure and nanostructure, which enable a remarkably water-resistant and self-cleaning surface. Insect wings are used as a basis for engineering biomimetic materials; however, the material properties of these nanostructures such as local elastic modulus and adhesion are poorly understood. We studied the wings of the Canton-S strain of Drosophila melanogaster (hereafter referred to as Drosophila) with atomic force microscopy (AFM) to quantify the local material properties of Drosophila wing surface nanostructures. The wings are found to have a hierarchical structure of 10-20 μm long, 0.5-1 μm diameter hair, and at a much smaller scale, 100 nm diameter and 30-60 nm high bumps. The local properties of these nanoscale bumps were studied under ambient and dry conditions with force-volume AFM. The wing membrane was found to have a elastic modulus on the order of 1000 MPa and the work of adhesion between the probe and wing membrane surface was found to be on the order of 100 mJ/m2, these properties are the same order of magnitude as common thermoplastic polymers such as polyethylene. The difference in work of adhesion between the nanoscale bump and membrane does not change significantly between ambient (relative humidity of 30%) or dry conditions. This suggests that the nanoscale bumps and the surrounding membrane are chemically similar and only work to increase hydrophobicity though surface roughening or the geometric lotus effect.

  19. Plasma Membrane and Nuclear Localization of G Protein–coupled Receptor Kinase 6A

    PubMed Central

    Jiang, Xiaoshan; Benovic, Jeffrey L.

    2007-01-01

    G protein–coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization. PMID:17538017

  20. Plasma membrane and nuclear localization of G protein coupled receptor kinase 6A.

    PubMed

    Jiang, Xiaoshan; Benovic, Jeffrey L; Wedegaertner, Philip B

    2007-08-01

    G protein-coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.

  1. Buckling of Actin-Coated Membranes under Application of a Local Force

    NASA Astrophysics Data System (ADS)

    Helfer, E.; Harlepp, S.; Bourdieu, L.; Robert, J.; Mackintosh, F. C.; Chatenay, D.

    2001-08-01

    The mechanical properties of composite membranes obtained by self-assembly of actin filaments with giant fluid vesicles are studied by micromanipulation with optical tweezers. These complexes exhibit typical mechanical features of a solid shell, including a finite in-plane shear elastic modulus (~10-6 N/m). A buckling instability is observed when a localized force of the order of 0.5 pN is applied perpendicular to the membrane plane. Although predicted for polymerized vesicles, this is the first evidence of such an instability.

  2. Buckling of actin-coated membranes under application of a local force.

    PubMed

    Helfer, E; Harlepp, S; Bourdieu, L; Robert, J; MacKintosh, F C; Chatenay, D

    2001-08-20

    The mechanical properties of composite membranes obtained by self-assembly of actin filaments with giant fluid vesicles are studied by micromanipulation with optical tweezers. These complexes exhibit typical mechanical features of a solid shell, including a finite in-plane shear elastic modulus ( approximately 10(-6) N/m). A buckling instability is observed when a localized force of the order of 0.5 pN is applied perpendicular to the membrane plane. Although predicted for polymerized vesicles, this is the first evidence of such an instability.

  3. Guanosine 5'-[gamma-thio]triphosphate induces membrane localization of cytosol-independent phospholipase D activity in a cell-free system from U937 promonocytic leucocytes.

    PubMed Central

    Kusner, D J; Dubyak, G R

    1994-01-01

    Activation of phospholipase D (PLD) in phagocytic leucocytes requires protein components present in both the plasma membrane and the cytosol, but the catalytic and regulatory factors are not fully defined. We have characterized the effect of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the subcellular requirements for reconstitution of PLD activity, using a cell-free system from U937 human promonocytic leucocytes. Incubation of permeabilized cells with 100 microM GTP[S] resulted in a membrane-localized PLD activity which was independent of added cytosol. The PLD activity of membranes from GTP[S]-treated cells was 7-fold greater than the basal activity of control membranes, and could be further augmented by the addition of ATP. This was the first demonstration of a stable agonist-regulated PLD activity in membranes from phagocytic leucocytes which was quantitatively comparable with that seen in a fully reconstituted system. Cytosol from GTP[S]-treated cells had a decreased capacity to support PLD activation, consistent with GTP[S]-induced depletion of a factor essential for reconstitution of PLD activity. Incubation of isolated membrane and cytosol with GTP[S] also resulted in a cytosol-independent PLD activity in the re-isolated membranes. The effect of GTP[S] could be mimicked by guanosine 5'-[beta gamma-imido]triphosphate, but not by aluminium fluoride, consistent with the involvement of a low-molecular-mass GTP-binding protein(s). Incubation of isolated subcellular fractions with GTP[S], followed by removal of unbound nucleotide, suggested that at least one of the GTP-binding proteins involved in the membrane localization of PLD activity was itself present in the membrane fraction. These data were consistent with a model in which activation of GTP-binding protein(s) resulted in the stable assembly of an active PLD signalling complex at the membrane surface. PMID:7998984

  4. A novel crystallization method for visualizing the membrane localization of potassium channels.

    PubMed Central

    Lopatin, A N; Makhina, E N; Nichols, C G

    1998-01-01

    The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel. PMID:9591643

  5. Local Laser Strengthening of Steel Sheets for Load Adapted Component Design in Car Body Structures

    NASA Astrophysics Data System (ADS)

    Jahn, Axel; Heitmanek, Marco; Standfuss, Jens; Brenner, Berndt; Wunderlich, Gerd; Donat, Bernd

    The current trend in car body construction concerning light weight design and car safety improvement increasingly requires an adaption of the local material properties on the component load. Martensitic hardenable steels, which are typically used in car body components, show a significant hardening effect, for instance in laser welded seams. This effect can be purposefully used as a local strengthening method. For several steel grades the local strengthening, resulting from a laser remelting process was investigated. The strength in the treated zone was determined at crash relevant strain rates. A load adapted design of complex reinforcement structures was developed for compression and bending loaded tube samples, using numerical simulation of the deformation behavior. Especially for bending loaded parts, the crash energy absorption can be increased significantly by local laser strengthening.

  6. Distribution and origin of the basement membrane component perlecan in rat liver and primary hepatocyte culture.

    PubMed Central

    Rescan, P. Y.; Loréal, O.; Hassell, J. R.; Yamada, Y.; Guillouzo, A.; Clément, B.

    1993-01-01

    Basement membranes contain three major components (ie collagen IV, laminin, and the heparan sulfate proteoglycan termed perlecan). Although the distribution and origin of both collagen IV and laminin have been well documented in the liver, perlecan has been poorly investigated, so far. We have studied the distribution and cellular origin of perlecan in rat livers in various conditions as well as in hepatocyte primary culture. By immunolocalization in both adult and 18-day-old fetal liver, perlecan was found in portal spaces, around central veins, and throughout the lobule. Immunoelectron microscopy revealed its presence at the level of basement membranes surrounding bile ducts and blood vessels, and in the space of Disse discontinuously interacting with hepatocyte microvilli. Precursors of perlecan were detected in the rough endoplasmic reticulum of bile duct cells and both vascular and sinusoidal endothelial cells. Both hepatocytes and Ito cells were negative. Northern-blot analysis confirmed the lack of appreciable expression of perlecan in hepatocytes isolated from either fetal or adult livers. In 18-month-diethylnitrosamine-treated rat liver, perlecan was abundant in neoplastic nodules. Electron microscopic investigation revealed an almost continuous layer of perlecan in the space of Disse and intracellular staining in sinusoidal endothelial cells, only. Perlecan mRNAs were detectable in malignant nodules, and absent in hepatocytes from nontumorous areas. Hepatocytes expressed high levels of perlecan mRNAs only when put in culture. This expression was reduced in conditions that allow improvement of hepatocyte survival and function (ie addition of corticoids, dimethylsulfoxide or nicotinamide to the medium, or in coculture with liver epithelial cells from biliary origin). Immunolocalization by light and electron microscopy showed that deposition of the proteoglycan occurred in coculture, in basement membranelike structures located around hepatocyte cords. In

  7. Localized waves in three-component coupled nonlinear Schrödinger equation

    NASA Astrophysics Data System (ADS)

    Xu, Tao; Chen, Yong

    2016-09-01

    We study the generalized Darboux transformation to the three-component coupled nonlinear Schrödinger equation. First- and second-order localized waves are obtained by this technique. In first-order localized wave, we get the interactional solutions between first-order rogue wave and one-dark, one-bright soliton respectively. Meanwhile, the interactional solutions between one-breather and first-order rogue wave are also given. In second-order localized wave, one-dark-one-bright soliton together with second-order rogue wave is presented in the first component, and two-bright soliton together with second-order rogue wave are gained respectively in the other two components. Besides, we observe second-order rogue wave together with one-breather in three components. Moreover, by increasing the absolute values of two free parameters, the nonlinear waves merge with each other distinctly. These results further reveal the interesting dynamic structures of localized waves in the three-component coupled system. Project supported by the Global Change Research Program of China (Grant No. 2015CB953904), the National Natural Science Foundation of China (Grant Nos. 11275072 and 11435005), the Doctoral Program of Higher Education of China (Grant No. 20120076110024), the Network Information Physics Calculation of Basic Research Innovation Research Group of China (Grant No. 61321064), and Shanghai Collaborative Innovation Center of Trustworthy Software for Internet of Things, China (Grant No. ZF1213).

  8. Surface-Localized Spermidine Protects the Pseudomonas aeruginosa Outer Membrane from Antibiotic Treatment and Oxidative Stress

    PubMed Central

    Johnson, Lori; Mulcahy, Heidi; Kanevets, Uliana; Shi, Yan

    2012-01-01

    Extracellular DNA acts as a cation chelator and induces the expression of antibiotic resistance genes regulated by Mg2+ levels. Here we report the characterization of novel DNA-induced genes in Pseudomonas aeruginosa that are annotated as homologs of the spermidine synthesis genes speD (PA4773) and speE (PA4774). The addition of sublethal concentrations of DNA and membrane-damaging antibiotics induced expression of the genes PA4773 to PA4775, as shown using transcriptional lux fusions and quantitative RT-PCR. Exogenous polyamine addition prevented DNA- and peptide-mediated gene induction. Mutation of PA4774 resulted in an increased outer membrane (OM) susceptibility phenotype upon polymyxin B, CP10A, and gentamicin treatment. When the membrane-localized fluorescent probe C11-BODIPY581/591 was used as an indicator of peroxidation of membrane lipids, the PA4774::lux mutant demonstrated an increased susceptibility to oxidative membrane damage from H2O2 treatment. Addition of exogenous polyamines protected the membranes of the PA4774::lux mutant from polymyxin B and H2O2 treatment. Polyamines from the outer surface were isolated and shown to contain putrescine and spermidine by using high-performance liquid chromatography and mass spectrometry. The PA4774::lux mutant did not produce spermidine on the cell surface, but genetic complementation restored surface spermidine production as well as the antibiotic and oxidative stress resistance phenotypes of the membrane. We have identified new functions for spermidine on the cell surface and propose that polyamines are produced under Mg2+-limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage. PMID:22155771

  9. Pivotal Advance: Phospholipids determine net membrane surface charge resulting in differential localization of active Rac1 and Rac2.

    PubMed

    Magalhaes, Marco A O; Glogauer, Michael

    2010-04-01

    In this investigation, we used primary murine neutrophils to demonstrate that local changes in membrane phospholipid composition alter the net cytoplasmic membrane surface charge, which results in selective recruitment of Rac1 or Rac2 based on the net charge of their respective C-terminal domains. Murine neutrophils undergoing chemotaxis or carrying out phagocytosis were transfected with K-ras4B-derived membrane charge biosensors and lipid markers, which allowed us to simultaneously monitor the levels of PIP(2), PIP(3), and PS and net membrane charge of the newly developing phagosome membrane and plasma membrane. Our results indicate that the combination of PIP(2), PIP(3), and PS generates a high negative charge (-8) at the plasma membrane of actin-rich pseudopods, where active Rac1 preferentially localizes during phagosome formation. The lipid metabolism that occurs during phagosome maturation results in the localized depletion of PIP(2), PIP(3), and partial decrease in PS. This creates a moderately negative net charge that correlates with the localization of active Rac2. Conversely, the accumulation of PIP(3) at the leading-edge membrane during chemotaxis generates a polarized accumulation of negative charges that recruits Rac1. These results provide evidence that alterations in membrane lipid composition and inner-membrane surface charge are important elements for the recruitment of differentially charged proteins and localization of signaling pathways during phagocytosis and chemotaxis in neutrophils.

  10. Process for recycling components of a PEM fuel cell membrane electrode assembly

    DOEpatents

    Shore, Lawrence [Edison, NJ

    2012-02-28

    The membrane electrode assembly (MEA) of a PEM fuel cell can be recycled by contacting the MEA with a lower alkyl alcohol solvent which separates the membrane from the anode and cathode layers of the assembly. The resulting solution containing both the polymer membrane and supported noble metal catalysts can be heated under mild conditions to disperse the polymer membrane as particles and the supported noble metal catalysts and polymer membrane particles separated by known filtration means.

  11. Localization of Membrane Proteins in the Cyanobacterium Synechococcus sp. PCC7942 (Radial Asymmetry in the Photosynthetic Complexes).

    PubMed Central

    Sherman, D. M.; Troyan, T. A.; Sherman, L. A.

    1994-01-01

    Localization of membrane proteins in the cyanobacterium Synechococcus sp. PCC7942 was determined by transmission electron microscopy utilizing immunocytochemistry with cells prepared by freeze-substitution. This preparation procedure maintained cellular morphology and permitted detection of cellular antigens with high sensitivity and low background. Synechococcus sp. PCC7942 is a unicellular cyanobacterium with thylakoids organized in concentric layers toward the periphery of the cell. Cytochrome oxidase was localized almost entirely in the cytoplasmic membrane, whereas a carotenoprotein (P35) was shown to be a cell wall component. The major photosystem II (PSII) proteins (D1, D2 CP43, and CP47) were localized throughout the thylakoids. Proteins of the Cyt b6/f complex were found to have a similar distribution. Thylakoid luminal proteins, such as the Mn-stabilizing protein, were located primarily in the thylakoid, but a small, reproducible fraction was found in the outer compartment. The photosystem I (PSI) reaction center proteins and the ATP synthase proteins were found associated mostly with the outermost thylakoid and with the cytoplasmic membrane. These results indicated that the photosynthetic apparatus is not evenly distributed throughout the thylakoids. Rather, there is a radial asymmetry such that much of the PSI and the ATPase synthase is located in the outermost thylakoid. The relationship of this structure to the photosynthetic mechanism is discussed. It is suggested that the photosystems are separated because of kinetic differences between PSII and PSI, as hypothesized by H.-W. Trissl and C. Wilhelm (Trends Biochem Sci [1993] 18:415-419). PMID:12232325

  12. Differential expression of epithelial basement membrane components nidogens and perlecan in corneal stromal cells in vitro.

    PubMed

    Santhanam, Abirami; Torricelli, Andre A M; Wu, Jiahui; Marino, Gustavo K; Wilson, Steven E

    2015-01-01

    The purpose of this study was to examine the expression of corneal epithelial basement membrane (EBM) components in different corneal stromal cell types. In vitro model systems were used to explore the expression of EBM components nidogen-1, nidogen-2, and perlecan that are the primary components in the lamina lucida and the lamina densa that defectively regenerate in corneas with stromal opacity after in -9.0 D photorefractive keratectomy (PRK). Primary rabbit corneal stromal cells were cultured using varying serum concentrations and exogenous growth factors, including fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1, to optimize the growth of each cell type of interest. The expression of the keratocyte-specific marker keratocan and the myofibroblast-specific marker α-smooth muscle actin (α-SMA) were analyzed with real-time PCR, western blot, and immunocytochemical staining to evaluate the specificity of the cell types and select optimal conditions (high keratocan and low α-SMA for keratocytes; low keratocan and high α-SMA for myofibroblasts; low keratocan and low α-SMA for corneal fibroblasts). The expression of the EBM components nidogen-1, nidogen-2, and perlecan was evaluated in each corneal cell type using real-time PCR, immunostaining, and western blotting. In agreement with previous studies, serum-free DMEM was found to be optimal for keratocytes, DMEM with 10% serum and 40 ng/ml FGF-2 yielded the best marker profile for corneal fibroblasts, and DMEM with 1% serum and 2 ng/ml TGF-β1 was found to be optimal for myofibroblasts. Nidogen-1 and nidogen-2 mRNAs were highly expressed in keratocytes, whereas perlecan was highly expressed in myofibroblasts. In keratocytes, nidogen-2 and perlecan proteins were expressed predominantly in intracellular compartments, whereas in myofibroblasts expression of both EBM components was observed diffusely throughout the cell. Although the perlecan mRNA levels were high in the myofibroblasts, the

  13. Immunohistochemical features of proliferative marker and basement membrane components of two feline inductive odontogenic tumours.

    PubMed

    Sakai, Hiroki; Mori, Takashi; Iida, Tsuneyoshi; Tokuma, Yanai; Maruo, Kouji; Masegi, Toshiaki

    2008-07-01

    Feline inductive odontogenic tumour (FIOT) is a rare and interesting odontogenic neoplasm in which the odontogenic epithelium has inductive potential to form aggregated foci of dental pulp-like mesenchymal cells. Two male cats aged 11 and 10 months presented with nasal swelling and a left maxillary mass. Histopathologically, the masses consisted of non-encapsulated invasive neoplasms exhibiting proliferation of epithelial and mesenchymal components with local infiltration into the maxillary bone in both cases. The epithelial component formed islands, anastomosing strands, and solid sheets of polygonal epithelial cells. Occasionally, these cells formed circular aggregates, resembling the cap stage of odontogenesis. Type IV collagen and laminin were constantly positive around the foci of epithelial cells, and Ki-67 positive indices were extremely low; therefore, these findings consistent with the benign clinical presentation of FIOT.

  14. Influence of Global and Local Membrane Curvature on Mechanosensitive Ion Channels: A Finite Element Approach

    PubMed Central

    Bavi, Omid; Cox, Charles D.; Vossoughi, Manouchehr; Naghdabadi, Reza; Jamali, Yousef; Martinac, Boris

    2016-01-01

    Mechanosensitive (MS) channels are ubiquitous molecular force sensors that respond to a number of different mechanical stimuli including tensile, compressive and shear stress. MS channels are also proposed to be molecular curvature sensors gating in response to bending in their local environment. One of the main mechanisms to functionally study these channels is the patch clamp technique. However, the patch of membrane surveyed using this methodology is far from physiological. Here we use continuum mechanics to probe the question of how curvature, in a standard patch clamp experiment, at different length scales (global and local) affects a model MS channel. Firstly, to increase the accuracy of the Laplace’s equation in tension estimation in a patch membrane and to be able to more precisely describe the transient phenomena happening during patch clamping, we propose a modified Laplace’s equation. Most importantly, we unambiguously show that the global curvature of a patch, which is visible under the microscope during patch clamp experiments, is of negligible energetic consequence for activation of an MS channel in a model membrane. However, the local curvature (RL < 50) and the direction of bending are able to cause considerable changes in the stress distribution through the thickness of the membrane. Not only does local bending, in the order of physiologically relevant curvatures, cause a substantial change in the pressure profile but it also significantly modifies the stress distribution in response to force application. Understanding these stress variations in regions of high local bending is essential for a complete understanding of the effects of curvature on MS channels. PMID:26861405

  15. Influence of Global and Local Membrane Curvature on Mechanosensitive Ion Channels: A Finite Element Approach.

    PubMed

    Bavi, Omid; Cox, Charles D; Vossoughi, Manouchehr; Naghdabadi, Reza; Jamali, Yousef; Martinac, Boris

    2016-02-05

    Mechanosensitive (MS) channels are ubiquitous molecular force sensors that respond to a number of different mechanical stimuli including tensile, compressive and shear stress. MS channels are also proposed to be molecular curvature sensors gating in response to bending in their local environment. One of the main mechanisms to functionally study these channels is the patch clamp technique. However, the patch of membrane surveyed using this methodology is far from physiological. Here we use continuum mechanics to probe the question of how curvature, in a standard patch clamp experiment, at different length scales (global and local) affects a model MS channel. Firstly, to increase the accuracy of the Laplace's equation in tension estimation in a patch membrane and to be able to more precisely describe the transient phenomena happening during patch clamping, we propose a modified Laplace's equation. Most importantly, we unambiguously show that the global curvature of a patch, which is visible under the microscope during patch clamp experiments, is of negligible energetic consequence for activation of an MS channel in a model membrane. However, the local curvature (RL < 50) and the direction of bending are able to cause considerable changes in the stress distribution through the thickness of the membrane. Not only does local bending, in the order of physiologically relevant curvatures, cause a substantial change in the pressure profile but it also significantly modifies the stress distribution in response to force application. Understanding these stress variations in regions of high local bending is essential for a complete understanding of the effects of curvature on MS channels.

  16. Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

  17. Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

  18. Membrane and Capillary Components of Lung Diffusion in Infants with Bronchopulmonary Dysplasia

    PubMed Central

    Chang, Daniel V.; Assaf, Santiago J.; Tiller, Christina J.; Kisling, Jeffrey A.

    2016-01-01

    Rationale: Autopsied lungs of infants with bronchopulmonary dysplasia (BPD) demonstrate impaired alveolar development with larger and fewer alveoli, which is consistent with our previous physiologic findings of lower pulmonary diffusing capacity of the lung for carbon monoxide (DlCO) in infants and toddlers with BPD compared with healthy controls born at full term (FT). However, it is not known whether the decreased DlCO in infants with BPD results from a reduction in both components of DlCO: pulmonary membrane diffusing capacity (Dm) and Vc. Objectives: We hypothesized that impairment of alveolar development in BPD results in a decrease in both Dm and Vc components of DlCO but that the Dm/Vc ratio would not differ between the BPD and FT groups. Methods: DlCO was measured under conditions of room air and high inspired oxygen (90%), which enabled Dm and Vc to be calculated. Measurements and Main Results: Dm and Vc increased with increasing body length; however, infants with BPD had significantly lower Dm and Vc than FT subjects after adjustment for race, sex, body length, and corrected age. In contrast to Dm and Vc, the Dm/Vc ratio remained constant with increasing body length and did not differ for infants with BPD and FT subjects. Conclusions: Our findings are consistent with infants with BPD having impaired alveolar development with fewer but larger alveoli, as well as a reduced Vc. PMID:26566056

  19. Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains

    PubMed Central

    Bücherl, Christoph A; Jarsch, Iris K; Schudoma, Christian; Segonzac, Cécile; Mbengue, Malick; Robatzek, Silke; MacLean, Daniel; Ott, Thomas; Zipfel, Cyril

    2017-01-01

    Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains. DOI: http://dx.doi.org/10.7554/eLife.25114.001 PMID:28262094

  20. Local Entropy Production Rates in a Polymer Electrolyte Membrane Fuel Cell

    NASA Astrophysics Data System (ADS)

    Siemer, Marc; Marquardt, Tobias; Valadez Huerta, Gerardo; Kabelac, Stephan

    2017-01-01

    A modeling study on a polymer electrolyte membrane fuel cell by means of non-equilibrium thermodynamics is presented. The developed model considers a one-dimensional cell in steady-state operation. The temperature, concentration and electric potential profiles are calculated for every domain of the cell. While the gas diffusion and the catalyst layers are calculated with established classical modeling approaches, the transport processes in the membrane are calculated with the phenomenological equations as dictated by the non-equilibrium thermodynamics. This approach is especially instructive for the membrane as the coupled transport mechanisms are dominant. The needed phenomenological coefficients are approximated on the base of conventional transport coefficients. Knowing the fluxes and their intrinsic corresponding forces, the local entropy production rate is calculated. Accordingly, the different loss mechanisms can be detected and quantified, which is important for cell and stack optimization.

  1. Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization.

    PubMed

    Ishikawa, Kazuya; Hashimoto, Masayoshi; Yusa, Akira; Koinuma, Hiroaki; Kitazawa, Yugo; Netsu, Osamu; Yamaji, Yasuyuki; Namba, Shigetou

    2017-06-01

    Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD.

  2. Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization

    PubMed Central

    Ishikawa, Kazuya; Hashimoto, Masayoshi; Yusa, Akira; Koinuma, Hiroaki; Kitazawa, Yugo; Netsu, Osamu; Yamaji, Yasuyuki; Namba, Shigetou

    2017-01-01

    Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD. PMID:28640879

  3. Reversed-phase liquid chromatographic retention and membrane activity relationships of local anesthetics.

    PubMed

    Tsuchiya, Hironori; Mizogami, Maki; Takakura, Ko

    2005-05-06

    The chromatographic retention and membrane activity relationships of local anesthetics were studied to address the possible mechanisms for structure specificity and inflammation-associated decrease of their effects. Five representative drugs (3 mM for each) were reacted with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes in 25 mM potassium phosphate buffer (pH 5.9-7.9, containing 100 mM NaCl and 0.1 mM EDTA) for 10 min at 37 degrees C and the membrane fluidity changes were analyzed by measuring fluorescence polarization with 1,6-diphenyl-1,3,5-hexatriene. Their capacity factors were determined on octadecyl-, octyl- and phenyl-bonded silica columns with a mobile phase consisting of 25 mM potassium phosphate buffer (pH 5.9-7.9, containing 100 mM NaCl and 0.1 mM EDTA)-methanol (30:70, v/v) at a flow rate of 1.0 ml/min and at a column temperature of 37 degrees C and diode-array detection. Mepivacaine, prilocaine, lidocaine, ropivacaine and bupivacaine fluidized membranes in increasing order of intensity, which agreed with their clinical potency. The relative degree of membrane fluidization correlated with that of retention on an octadecyl stationary phase more significantly than the other phases. Both membrane-fluidizing effects and capacity factors decreased by lowering the reaction and mobile phase pH, being consistent with the hypothesis that anesthetic potency is reduced in inflammation because of tissue acidity. Reversed-phase liquid chromatography appears to be useful for estimating the structure-specific and pH-dependent membrane-fluidizing effects of local anesthetics.

  4. Performance of Water Recirculation Loop Maintenance Components for the Advanced Spacesuit Water Membrane Evaporator

    NASA Technical Reports Server (NTRS)

    Rector, Tony; Peyton, Barbara M.; Steele, John W.; Makinen, Janice; Bue, Grant C.; Campbell, Colin

    2014-01-01

    Water loop maintenance components to maintain the water quality of the Advanced Spacesuit Water Membrane Evaporation (SWME) water recirculation loop have undergone a comparative performance evaluation with a second SWME water recirculation loop with no water quality maintenance. Results show the benefits of periodic water maintenance. The SWME is a heat rejection device under development at the NASA Johnson Space Center to perform thermal control for advanced spacesuits. One advantage to this technology is the potential for a significantly greater degree of tolerance to contamination when compared to the existing Sublimator technology. The driver for the evaluation of water recirculation maintenance components was to further enhance this advantage through the leveraging of fluid loop management lessons learned from the International Space Station (ISS). A bed design that was developed for a UTAS military application, and considered for a potential ISS application with the Urine Processor Assembly, provided a low pressure drop means for water maintenance in a recirculation loop. The bed design is coupled with high capacity ion exchange resins, organic adsorbents, and a cyclic methodology developed for the Extravehicular Mobility Unit (EMU) Transport Water loop. The maintenance cycle included the use of a biocide delivery component developed for ISS to introduce a biocide in a microgravity compatible manner for the Internal Active Thermal Control System (IATCS). The leveraging of these water maintenance technologies to the SWME recirculation loop is a unique demonstration of applying the valuable lessons learned on the ISS to the next generation of manned spaceflight Environmental Control and Life Support System (ECLSS) hardware.

  5. Performance of Water Recirculation Loop Maintentance Components for the Advanced Spacesuit Water Membrane Evaporator

    NASA Technical Reports Server (NTRS)

    Rector, Tony; Peyton, Barbara; Steele, John W.; Bue, Grant C.; Campbell, Colin; Makinen, Janice

    2014-01-01

    Water loop maintenance components to maintain the water quality of the Advanced Spacesuit Water Membrane Evaporation (SWME) water recirculation loop have undergone a comparative performance evaluation with a second SWME water recirculation loop with no water quality maintenance. Results show the benefits of periodic water maintenance. The SWME is a heat rejection device under development at the NASA Johnson Space Center to perform thermal control for advanced spacesuits. One advantage to this technology is the potential for a significantly greater degree of tolerance to contamination when compared to the existing Sublimator technology. The driver for the evaluation of water recirculation maintenance components was to further enhance this advantage through the leveraging of fluid loop management lessonslearned from the International Space Station (ISS). A bed design that was developed for a UTAS military application, and considered for a potential ISS application with the Urine Processor Assembly, provided a low pressure drop means for water maintenance in a recirculation loop. The bed design is coupled with high capacity ion exchange resins, organic adsorbents, and a cyclic methodology developed for the Extravehicular Mobility Unit (EMU) Transport Water loop. The maintenance cycle included the use of a biocide delivery component developed for ISS to introduce a biocide in a microgravity-compatible manner for the Internal Active Thermal Control System (IATCS). The leveraging of these water maintenance technologies to the SWME recirculation loop is a unique demonstration of applying the valuable lessons learned on the ISS to the next generation of manned spaceflight Environmental Control and Life Support System (ECLSS) hardware.

  6. Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth

    PubMed Central

    1987-01-01

    We have examined the movements, composition, and cellular origin of phase-dense varicosities in cultures of chick sympathetic and sensory neurons. These organelles are variable in diameter (typically between 0.2 and 2 microns) and undergo saltatory movements both towards and away from the neuronal cell body. Their mean velocities vary inversely with the size of the organelle and are greater in the retrograde than the anterograde direction. Organelles stain with the lipophilic dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine and with antibodies to cytoskeletal components. In cultures double-stained with antibodies to alpha-tubulin and 70-kD neurofilament protein (NF-L), approximately 40% of the organelles stain for tubulin, 30% stain for NF- L, 10% stain for both tubulin and NF-L, and 40% show no staining with either antibody. The association of cytoskeletal proteins with the organelles shows that these proteins are able to move by a form of rapid axonal transport. Under most culture conditions the predominant direction of movement is towards the cell body, suggesting that the organelles are produced at or near the growth cone. Retrograde movements continue in culture medium lacking protein or high molecular mass components and increase under conditions in which the advance of the growth cone is arrested. There is a fourfold increase in the number of organelles moving retrogradely in neurites that encounter a substratum-associated barrier to elongation; retrograde movements increase similarly in cultures exposed to cytochalasin at levels known to block growth cone advance. No previously described organelle shows behavior coordinated with axonal growth in this way. We propose that the organelles contain membrane and cytoskeletal components that have been delivered to the growth cone, by slow or fast anterograde transport, in excess of the amounts required to synthesize more axon. In view of their rapid mobility and variable contents, we suggest that they

  7. Local Anesthetics and Antipsychotic Phenothiazines Interact Nonspecifically with Membranes and Inhibit Hexose Transporters in Yeast

    PubMed Central

    Uesono, Yukifumi; Toh-e, Akio; Kikuchi, Yoshiko; Araki, Tomoyuki; Hachiya, Takushi; Watanabe, Chihiro K.; Noguchi, Ko; Terashima, Ichiro

    2016-01-01

    Action mechanisms of anesthetics remain unclear because of difficulty in explaining how structurally different anesthetics cause similar effects. In Saccharomyces cerevisiae, local anesthetics and antipsychotic phenothiazines induced responses similar to those caused by glucose starvation, and they eventually inhibited cell growth. These drugs inhibited glucose uptake, but additional glucose conferred resistance to their effects; hence, the primary action of the drugs is to cause glucose starvation. In hxt0 strains with all hexose transporter (HXT) genes deleted, a strain harboring a single copy of HXT1 (HXT1s) was more sensitive to tetracaine than a strain harboring multiple copies (HXT1m), which indicates that quantitative reduction of HXT1 increases tetracaine sensitivity. However, additional glucose rather than the overexpression of HXT1/2 conferred tetracaine resistance to wild-type yeast; therefore, Hxts that actively transport hexoses apparently confer tetracaine resistance. Additional glucose alleviated sensitivity to local anesthetics and phenothiazines in the HXT1m strain but not the HXT1s strain; thus, the glucose-induced effects required a certain amount of Hxt1. At low concentrations, fluorescent phenothiazines were distributed in various membranes. At higher concentrations, they destroyed the membranes and thereby delocalized Hxt1-GFP from the plasma membrane, similar to local anesthetics. These results suggest that the aforementioned drugs affect various membrane targets via nonspecific interactions with membranes. However, the drugs preferentially inhibit the function of abundant Hxts, resulting in glucose starvation. When Hxts are scarce, this preference is lost, thereby mitigating the alleviation by additional glucose. These results provide a mechanism that explains how different compounds induce similar effects based on lipid theory. PMID:26757771

  8. Dynamics of Crowded Vesicles: Local and Global Responses to Membrane Composition

    PubMed Central

    Holdbrook, Daniel A.; Huber, Roland G.; Piggot, Thomas J.; Bond, Peter J.; Khalid, Syma

    2016-01-01

    The bacterial cell envelope is composed of a mixture of different lipids and proteins, making it an inherently complex organelle. The interactions between integral membrane proteins and lipids are crucial for their respective spatial localization within bacterial cells. We have employed microsecond timescale coarse-grained molecular dynamics simulations of vesicles of varying sizes and with a range of protein and lipid compositions, and used novel approaches to measure both local and global system dynamics, the latter based on spherical harmonics analysis. Our results suggest that both hydrophobic mismatch, enhanced by embedded membrane proteins, and curvature based sorting, due to different modes of undulation, may drive assembly in vesicular systems. Interestingly, the modes of undulation of the vesicles were found to be altered by the specific protein and lipid composition of the vesicle. Strikingly, lipid dynamics were shown to be coupled to proteins up to 6 nm from their surface, a substantially larger distance than has previously been observed, resulting in multi-layered annular rings enriched with particular types of phospholipid. Such large protein-lipid complexes may provide a mechanism for long-range communication. Given the complexity of bacterial membranes, our results suggest that subtle changes in lipid composition may have major implications for lipid and protein sorting under a curvature-based membrane-sorting model. PMID:27310814

  9. The X-ray Emitting Components towards l = 111 deg: The Local Hot Bubble and Beyond

    NASA Technical Reports Server (NTRS)

    Kuntz, K. D.; Snowden, S. L.

    2006-01-01

    We have obtained an XMM-Newton spectrum of the diffuse X-ray emission towards (l, b) = (111.14,1.11), a line of sight with a relatively simple distribution of absorbing clouds; > 9 x 10(exp 19)/sq cm at R>170 pc, a 6 x 10(exp 21)/sq cm molecular cloud at 2.5-3.3 kpc, and a total column of 1.2 x 10(exp 22)/sq cm. We find that the analysis of the XMM-Newton spectrum in conjunction with the RASS spectral energy distribution for the same direction requires three thermal components to be well fit: a "standard" Local Hot Bubble component with kT = 0.089, a component beyond the molecular cloud with kT = 0.59, and a component before the molecular cloud with kT = 0.21. The strength of the O VII 0.56 keV line from the Local Hot Bubble, 2.1+/-0.7 photons/sq cm/s/sr, is consistent with other recent measures. The 0.21 keV component has an emission measure of 0.0022+/-0.0006 pc and is not localized save as diffuse emission within the Galactic plane; it is the best candidate for a pervasive hot medium. The spatial separation of the approx. 0.2 keV component from the approx. 0.6 keV component suggests that the spectral decompositions of the emission from late-type spiral disks found in the literature do represent real temperature components rather than reflecting more complex temperature distributions.

  10. Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes

    PubMed Central

    1985-01-01

    Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000- 43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending. PMID:3884630

  11. Electricity generation and local ion ordering induced by cation-controlled selective anion transportation through graphene oxide membranes

    NASA Astrophysics Data System (ADS)

    Sun, Pengzhan; Deng, Hui; Zheng, Feng; Wang, Kunlin; Zhong, Minlin; Zhang, Yingjiu; Kang, Feiyu; Zhu, Hongwei

    2014-12-01

    A cation-controlled selective anion transportation through graphene oxide (GO) membranes is demonstrated in this work. The results reveal that the trans-membrane transport of different anions can be modulated by the corresponding cations. The diverse interactions among anions, cations, and the negatively charged GO membranes are responsible for selective anion permeation through GO membranes. During the ion penetration, electrical potential differences can be generated across drain and source as well as across GO membranes; based on this, the ion distributions around GO membranes can be determined. The results indicate that local ion ordering can be achieved by GO membranes. Interestingly, for the cases of KNO3, Ca(NO3)2, and Ba(NO3)2, alternate aggregations of metallic cations and NO3- anions can be formed around GO membranes, demonstrating the fantastic ability of these membranes for ordering the ions locally in solutions. In addition, based on the electrical potential differences generated by different salts, chlorides are demonstrated to be ideal sources for efficient practical electricity production compared to sulfates and nitrates, while the different voltage signals generated can be used to identify different source solutions for liquid sensing applications. These results indicate that GO membranes can find potential applications in membrane separation, energy generation, ion recognition, and local ion organizing.

  12. Molecular counting of membrane receptor subunits with single-molecule localization microscopy

    NASA Astrophysics Data System (ADS)

    Krüger, Carmen; Fricke, Franziska; Karathanasis, Christos; Dietz, Marina S.; Malkusch, Sebastian; Hummer, Gerhard; Heilemann, Mike

    2017-02-01

    We report on quantitative single-molecule localization microscopy, a method that next to super-resolved images of cellular structures provides information on protein copy numbers in protein clusters. This approach is based on the analysis of blinking cycles of single fluorophores, and on a model-free description of the distribution of the number of blinking events. We describe the experimental and analytical procedures, present cellular data of plasma membrane proteins and discuss the applicability of this method.

  13. Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria.

    PubMed

    Andersen, C

    2003-01-01

    For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 A into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system.

  14. Plasmodium falciparum FIKK kinase members target distinct components of the erythrocyte membrane.

    PubMed

    Nunes, Marta C; Okada, Mami; Scheidig-Benatar, Christine; Cooke, Brian M; Scherf, Artur

    2010-07-23

    Modulation of infected host cells by intracellular pathogens is a prerequisite for successful establishment of infection. In the human malaria parasite Plasmodium falciparum, potential candidates for erythrocyte remodelling include the apicomplexan-specific FIKK kinase family (20 members), several of which have been demonstrated to be transported into the erythrocyte cytoplasm via Maurer's clefts. In the current work, we have knocked out two members of this gene family (Pf fikk7.1 and Pf fikk12), whose products are localized at the inner face of the erythrocyte membrane. Both mutant parasite lines were viable and erythrocytes infected with these parasites showed no detectable alteration in their ability to adhere in vitro to endothelial receptors such as chondroitin sulfate A and CD36. However, we observed sizeable decreases in the rigidity of infected erythrocytes in both knockout lines. Mutant parasites were further analyzed using a phospho-proteomic approach, which revealed distinct phosphorylation profiles in ghost preparations of infected erythrocytes. Knockout parasites showed a significant reduction in the level of phosphorylation of a protein of approximately 80 kDa for FIKK12-KO in trophozoite stage and a large protein of about 300 kDa for FIKK7.1-KO in schizont stage. Our results suggest that FIKK members phosphorylate different membrane skeleton proteins of the infected erythrocyte in a stage-specific manner, inducing alterations in the mechanical properties of the parasite-infected red blood cell. This suggests that these host cell modifications may contribute to the parasites' survival in the circulation of the human host.

  15. Plasmodium falciparum FIKK Kinase Members Target Distinct Components of the Erythrocyte Membrane

    PubMed Central

    Scheidig-Benatar, Christine; Cooke, Brian M.; Scherf, Artur

    2010-01-01

    Background Modulation of infected host cells by intracellular pathogens is a prerequisite for successful establishment of infection. In the human malaria parasite Plasmodium falciparum, potential candidates for erythrocyte remodelling include the apicomplexan-specific FIKK kinase family (20 members), several of which have been demonstrated to be transported into the erythrocyte cytoplasm via Maurer's clefts. Methodology In the current work, we have knocked out two members of this gene family (Pf fikk7.1 and Pf fikk12), whose products are localized at the inner face of the erythrocyte membrane. Both mutant parasite lines were viable and erythrocytes infected with these parasites showed no detectable alteration in their ability to adhere in vitro to endothelial receptors such as chondroitin sulfate A and CD36. However, we observed sizeable decreases in the rigidity of infected erythrocytes in both knockout lines. Mutant parasites were further analyzed using a phospho-proteomic approach, which revealed distinct phosphorylation profiles in ghost preparations of infected erythrocytes. Knockout parasites showed a significant reduction in the level of phosphorylation of a protein of approximately 80 kDa for FIKK12-KO in trophozoite stage and a large protein of about 300 kDa for FIKK7.1-KO in schizont stage. Conclusions Our results suggest that FIKK members phosphorylate different membrane skeleton proteins of the infected erythrocyte in a stage-specific manner, inducing alterations in the mechanical properties of the parasite-infected red blood cell. This suggests that these host cell modifications may contribute to the parasites' survival in the circulation of the human host. PMID:20668526

  16. Localization of the event-related potential novelty response as defined by principal components analysis.

    PubMed

    Dien, Joseph; Spencer, Kevin M; Donchin, Emanuel

    2003-10-01

    Recent research indicates that novel stimuli elicit at least two distinct components, the Novelty P3 and the P300. The P300 is thought to be elicited when a context updating mechanism is activated by a wide class of deviant events. The functional significance of the Novelty P3 is uncertain. Identification of the generator sources of the two components could provide additional information about their functional significance. Previous localization efforts have yielded conflicting results. The present report demonstrates that the use of principal components analysis (PCA) results in better convergence with knowledge about functional neuroanatomy than did previous localization efforts. The results are also more convincing than that obtained by two alternative methods, MUSIC-RAP and the Minimum Norm. Source modeling on 129-channel data with BESA and BrainVoyager suggests the P300 has sources in the temporal-parietal junction whereas the Novelty P3 has sources in the anterior cingulate.

  17. Separation of the global and local components in functional near-infrared spectroscopy signals using principal component spatial filtering.

    PubMed

    Zhang, Xian; Noah, Jack Adam; Hirsch, Joy

    2016-01-01

    Global systemic effects not specific to a task can be prominent in functional near-infrared spectroscopy (fNIRS) signals and the separation of task-specific fNIRS signals and global nonspecific effects is challenging due to waveform correlations. We describe a principal component spatial filter algorithm for separation of the global and local effects. The effectiveness of the approach is demonstrated using fNIRS signals acquired during a right finger-thumb tapping task where the response patterns are well established. Both the temporal waveforms and the spatial pattern consistencies between oxyhemoglobin and deoxyhemoglobin signals are significantly improved, consistent with the basic physiological basis of fNIRS signals and the expected pattern of activity associated with the task.

  18. The Equine Herpesvirus 1 US2 Homolog Encodes a Nonessential Membrane-Associated Virion Component

    PubMed Central

    Meindl, Alexandra; Osterrieder, Nikolaus

    1999-01-01

    Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 US2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 US2 protein specifically detected a protein with an Mr of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-Mr Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-Mr Us2 polypeptide. Irrespective of its size, the US2 protein was incorporated into virions. The EHV-1 US2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 US2 protein or to a truncated US2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 US2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the US2 protein in the viral envelope and plasma membrane of infected cells, a US2-negative RacL11 mutant (L11ΔUS2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a US2-repaired virus. After infection of BALB/c mice with L11ΔUS2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 US2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo. PMID:10074198

  19. The equine herpesvirus 1 Us2 homolog encodes a nonessential membrane-associated virion component.

    PubMed

    Meindl, A; Osterrieder, N

    1999-04-01

    Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 Us2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 Us2 protein specifically detected a protein with an Mr of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-Mr Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-Mr Us2 polypeptide. Irrespective of its size, the Us2 protein was incorporated into virions. The EHV-1 Us2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 Us2 protein or to a truncated Us2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 Us2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the Us2 protein in the viral envelope and plasma membrane of infected cells, a Us2-negative RacL11 mutant (L11DeltaUs2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a Us2-repaired virus. After infection of BALB/c mice with L11DeltaUs2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 Us2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.

  20. Comparative genomic analysis of evolutionarily conserved but functionally uncharacterized membrane proteins in archaea: Prediction of novel components of secretion, membrane remodeling and glycosylation systems.

    PubMed

    Makarova, Kira S; Galperin, Michael Y; Koonin, Eugene V

    2015-11-01

    A systematic comparative genomic analysis of all archaeal membrane proteins that have been projected to the last archaeal common ancestor gene set led to the identification of several novel components of predicted secretion, membrane remodeling, and protein glycosylation systems. Among other findings, most crenarchaea have been shown to encode highly diverged orthologs of the membrane insertase YidC, which is nearly universal in bacteria, eukaryotes, and euryarchaea. We also identified a vast family of archaeal proteins, including the C-terminal domain of N-glycosylation protein AglD, as membrane flippases homologous to the flippase domain of bacterial multipeptide resistance factor MprF, a bifunctional lysylphosphatidylglycerol synthase and flippase. Additionally, several proteins were predicted to function as membrane transporters. The results of this work, combined with our previous analyses, reveal an unexpected diversity of putative archaeal membrane-associated functional systems that remain to be functionally characterized. A more general conclusion from this work is that the currently available collection of archaeal (and bacterial) genomes could be sufficient to identify (almost) all widespread functional modules and develop experimentally testable predictions of their functions.

  1. Membrane-localized ubiquitin ligase ATL15 functions in sugar-responsive growth regulation in Arabidopsis.

    PubMed

    Aoyama, Shoki; Terada, Saki; Sanagi, Miho; Hasegawa, Yoko; Lu, Yu; Morita, Yoshie; Chiba, Yukako; Sato, Takeo; Yamaguchi, Junji

    2017-09-09

    Ubiquitin ligases play important roles in regulating various cellular processes by modulating the protein function of specific ubiquitination targets. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases that localize to membranes via their N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with several ATLs reported to be involved in regulating plant responses to environmental stresses. However, the functions of most ATLs remain unknown. This study, involving transcriptome database analysis, identifies ATL15 as a sugar responsive ATL gene in Arabidopsis. ATL15 expression was rapidly down-regulated in the presence of sugar. The ATL15 protein showed ubiquitin ligase activity in vitro and localized to plasma membrane and endomembrane compartments. Further genetic analyses demonstrated that the atl15 knockout mutants are insensitive to high glucose concentrations, whereas ATL15 overexpression depresses plant growth. In addition, endogenous glucose and starch amounts were reciprocally affected in the atl15 knockout mutants and the ATL15 overexpressors. These results suggest that ATL15 protein plays a significant role as a membrane-localized ubiquitin ligase that regulates sugar-responsive plant growth in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. 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

  3. Lateral organization of membranes and cell shapes.

    PubMed Central

    Markin, V S

    1981-01-01

    The relations among membrane structure, mechanical properties, and cell shape have been investigated. The fluid mosaic membrane models used contains several components that move freely in the membrane plane. These components interact with each other and determine properties of the membrane such as curvature and elasticity. A free energy equation is postulated for such a multicomponent membrane and the condition of free energy minimum is used to obtain differential equations relating the distribution of membrane components and the local membrane curvature. The force that moves membrane components along the membrane in a variable curvature field is calculated. A change in the intramembrane interactions can bring about phase separation or particle clustering. This, in turn, may strongly affect the local curvature. The numerical solution of the set of equations for the two dimensional case allows determination of the cell shape and the component distribution along the membrane. The model has been applied to describe certain erythrocytes shape transformations. PMID:7284547

  4. Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa.

    PubMed

    Moradali, M Fata; Ghods, Shirin; Rehm, Bernd H A

    2017-03-03

    The exopolysaccharide, alginate, produced by the opportunistic human pathogen Pseudomonas aeruginosa represents a survival advantage by contributing to formation of characteristic biofilms during infection. Membrane anchored proteins Alg8 (catalytic subunit) and Alg44 (co-polymerase) constitute the alginate polymerase which is being activated by the second messenger molecule c-di-GMP, but the mechanism of activation remains elusive. To shed light on the c-di-GMP mediated activation of alginate polymerization in vivo, an in silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops including H323 (loop A), T457 and E460 (loop B) surrounding the catalytic site in the predicted model. Activity of respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 co-polymerase constituted a stable dimer, with its periplasmic domains required for protein localization, alginate polymerization and modification. Superfolder GFP fusions of Alg8 and Alg44 showed a non-uniform, punctuate and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44 including their subcellular localization and distribution.IMPORTANCE The exopolysaccharide, alginate, is an important biofilm component of the opportunistic human pathogen P. aeruginosa and the principle

  5. Asparagine-linked oligosaccharides are localized to single extracytosolic segments in multi-span membrane glycoproteins.

    PubMed Central

    Landolt-Marticorena, C; Reithmeier, R A

    1994-01-01

    A comprehensive survey of mammalian multi-span (polytopic) membrane proteins showed that asparagine(N)-linked oligosaccharides are localized to single extracytosolic segments. In most membrane proteins this is because potential consensus sites for N-glycosylation (Asn-Xaa-Ser/Thr, X not equal to Pro) are not found in multiple extracytosolic segments. In functional proteins where consensus N-glycosylation sites are contained within more than one extracytosolic segment, only the first segment contains N-linked carbohydrate. An exception is the alpha-subunit of the Na+ channel, which consists of a duplicated structure containing two glycosylated segments. The average size of established N-glycosylated loops connecting two transmembrane segments is 62 residues, with the smallest glycosylated loop being 33 residues in size. N-glycosylated sites are more highly conserved than non-glycosylated (primarily cytosolic) sites and are more common toward the N-terminus of the membrane domain of multi-span membrane proteins. The optimal conditions for glycosylation of consensus sites within an extracytosolic domain of a multi-span membrane protein are (i) the acceptor site is well-spaced (greater than 10 residues) from the transmembrane domain, (ii) the loop is greater than 30 residues in size and (iii) the segment is the first in the protein to contain a suitable extracytosolic consensus site. The localization of N-linked oligosaccharide chains to a single protein segment suggests either glycosylation of multiple loops may compromise protein folding or function, or only a single polypeptide domain can be optimally glycosylated during biosynthesis in vivo. PMID:8068013

  6. Characterization of the key antigenic components of pertussis vaccine based on outer membrane vesicles.

    PubMed

    Ormazábal, Maximiliano; Bartel, Erika; Gaillard, María Emilia; Bottero, Daniela; Errea, Agustina; Zurita, M Eugenia; Moreno, Griselda; Rumbo, Martin; Castuma, Celina; Flores, Dario; Martín, María Julia; Hozbor, Daniela

    2014-10-21

    Pertussis has resurged during the last two decades in different countries. In particular in the 2010-2013 period large outbreaks were detected in US, Australia, UK and The Netherlands with significant mortality in infants. The epidemiological situation of pertussis points out the need to develop new vaccines and in this regard we previously developed a new vaccine based on outer membrane vesicles (OMVs) which have been shown to be safe and to induce protection in mice. Here we have further investigated the properties of OMVs vaccines; in particular we studied the contribution of pertussis toxin (PTx) and pertactin (Prn) in OMVs-mediated protection against pertussis. PTx-deficient OMVs and Prn-deficient OMVs were obtained from defective Bordetella pertussis mutants. The absence of PTx or Prn did compromise the protective capacity of the OMVs formulated as Tdap vaccine. Whereas the protective efficacy of the PTx-deficient OMVs in mice was comparable to Prn-deficient OMVs, the protective capacity of both of them was significantly impaired when it was compared with the wild type OMVs. Interestingly, using OMVs obtained from a B. pertussis strain which does not express any of the virulence factors but expresses the avirulent phenotype; we observed that the protective ability of such OMVs was lower than that of OMVs obtained from virulent B. pertussis phase. However, it was surprising that although the protective capacity of avirulent OMVs was lower, they were still protective in the used mice model. These results allow us to hypothesize that OMVs from avirulent phase shares protective components with all OMVs assayed. Using an immune proteomic strategy we identified some common components that could play an important role in protection against pertussis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Independent component analysis of localized resting-state functional magnetic resonance imaging reveals specific motor subnetworks.

    PubMed

    Sohn, William Seunghyun; Yoo, Kwangsun; Jeong, Yong

    2012-01-01

    Recent studies have shown that blood oxygen level-dependent low-frequency (<0.1 Hz) fluctuations (LFFs) during a resting-state exhibit a high degree of correlation with other regions that share cognitive function. Initial studies of resting-state network mapping have focused primarily on major networks such as the default mode network, primary motor, somatosensory, visual, and auditory networks. However, more specific or subnetworks, including those associated with specific motor functions, have yet to be properly addressed. We performed independent component analysis (ICA) in a specific target region of the brain, a process we name, "localized ICA." We demonstrated that when ICA is applied to localized fMRI data, it can be used to distinguish resting-state LFFs associated with specific motor functions (e.g., finger tapping, foot movement, or bilateral lip pulsing) in the primary motor cortex. These ICA components generated from localized data can then be used as functional regions of interest to map whole-brain connectivity. In addition, this method can be used to visualize inter-regional connectivity by expanding the localized region and identifying components that show connectivity between the two regions.

  8. Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia.

    PubMed

    Alexander, R Todd; Beggs, Megan R; Zamani, Reza; Marcussen, Niels; Frische, Sebastian; Dimke, Henrik

    2015-10-01

    Plasma membrane Ca(2+)-ATPases (PMCAs) participate in epithelial Ca(2+) transport and intracellular Ca(2+) signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial transient receptor potential vanilloid 5 Ca(2+) channel. We therefore hypothesized that Pmca4 plays a significant role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestines, whereas pan-specific Pmca antibodies detected Pmca1 in lateral membranes of enterocytes. In the kidney, Pmca4 showed broad localization to the distal nephron. In the mouse, expression was most abundant in segments coexpressing the epithelial ransient receptor potential vanilloid 5 Ca(2+) channel. Significant, albeit lower, expression was also evident in the region encompassing the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In the human kidney, a similar pattern of distribution was observed, with the highest PMCA4 expression in Na(+)-Cl(-) cotransporter-positive tubules. Electron microscopy demonstrated Pmca4 localization in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing to a housekeeping function of the pump in Ca(2+)-transporting epithelia. In conclusion, Pmca4 shows a divergent expression pattern in Ca(2+)-transporting epithelia, inferring diverse roles for this isoform not limited to transepithelial Ca(2+) transport.

  9. Some physico-chemical and serological properties of isolated protein components of red cell membranes

    PubMed Central

    Poulik, M. D.; Lauf, P. K.

    1969-01-01

    Exposure of human red cell ghosts to low pH (pH 2–2·2) for 20–24 hr and extraction with a biphasic system (butanol–water) at acid pH yielded a water-phase material which was separated by gel filtration into two major fractions. Fraction A is of glycoprotein nature having a molecular weight of approximately 200,000 and exhibiting A, M and N antigen activity. Fraction B is primarily protein in nature having a molecular weight of about 50,000 to 60,000, and is practically devoid of A and MN antigens. The materials isolated were found heterogeneous by electrophoresis in urea starch gels. Peak A and peak B differed significantly with respect to number, mobility, and intensity of the separated components. The physico-chemical and serological properties of the water phase and its sub-fractions are discussed with respect to structural make-up of erythrocyte membrane. ImagesFIG. 2FIG. 3 PMID:5786811

  10. Free fatty acids as a major component of the chlorosulfolipid membrane of Ochromonas danica

    SciTech Connect

    Winicov, I.

    1985-01-01

    This work is an attempt to determine whether or not free fatty acids are components of the natural membrane of Ochromonas danica. If the FFAs were artifacts, they would most likely have been produced during solvent extraction or during the procedure for flagellar detachment. Attempts to denature putative solvent-activated lipase(s) through exposure to boiling isopropanol or by crosslinking the flagella with glutaraldehyde prior to extraction failed to eliminate the free fatty acid fraction, nor to significantly alter its composition. Exposure of flagella to albumin resulted in the net transfer of FFAs to the supernatant phase, showing their presence is not caused by solvent activated lipolysis. Finally levels of labelled free fatty acids failed to rise as a function of time after deflagellation in cells grown in the presence of (10-/sup 14/C)-oleic acid. Acid hydrolysis of the total labelled lipid at elevated temperature increased the percentage of counts occurring as unesterified fatty acids (from 2.6% to 64.8%). This, taken together with a corresponding loss of the more polar labelled material (66.8% to 8.2%) indicates that some esterified lipids were present, but probably not broken down during the isolation procedure.

  11. Immunogenicity of Coxiella burnetii whole cells and their outer membrane components.

    PubMed

    Gajdosová, E; Kovácová, E; Toman, R; Skultéty, L; Lukácová, M; Kazár, J

    1994-12-01

    The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole cells (Cb I and Cb II) and their outer membrane components (OMC), i.e. phase I trichloroacetic acid extract (TCAE), phase I 29 K protein (PRO), phase I and II lipopolysaccharides (LPS I, LPS II), polysaccharides (PS I, PS II), and lipid A (LA I, LA II), were compared. The highest immune response was observed in BALB/c mice by Cb I in both humoral immunity and lymphocyte transformation assays, and in the protective effect as well. The immune response was also significant by Cb II, but their protective capacity was low. The OMC reacted variously. Only TCAE and PRO gave a high value of humoral immunity evaluated by the serological methods. All OMC reacted in the haemolytic plaque assay giving different responses. Lymphoproliferation of splenocytes was positive with all OMC using both Cb I and Cb II antigens with the exception of PS I and PS II in the case of Cb II antigen. The induction of protection against infectious Cb I was demonstrated after immunization with TCAE, PRO, and LPS I. Other OMC did not induce protection against this agent.

  12. Concentration gradient along scala tympani following the local application of gentamicin to the round window membrane

    PubMed Central

    Plontke, Stefan K.; Mynatt, Robert; Gill, Ruth M.; Borgmann, Stefan; Salt, Alec N.

    2008-01-01

    Objectives The distribution of gentamicin along the fluid spaces of the cochlea following local applications has never previously been demonstrated. Computer simulations have predicted that significant basal-apical concentration gradients might be expected and histological studies indicate that hair cell damage is greater at the base than at the apex following local gentamicin application. In the present study, gradients of gentamicin along the cochlea were measured. Methods A recently-developed method of sampling perilymph from the cochlear apex of guinea pigs was used, in which the samples represent fluid originating from different regions along scala tympani. Gentamicin concentration was determined in sequential apical samples which were taken following up to three hours of local application to the round window niche. Results Substantial gradients of gentamicin along the length of scala tympani were demonstrated and quantified, averaging more than 4000 times greater concentration at the base compared to the apex at the time of sampling. Peak concentrations and gradients for gentamicin varied considerably between animals, likely resulting from variations in round window membrane permeability and rates of perilymph flow. Conclusions The large gradients for gentamicin demonstrated here in guinea pigs account for how it is possible to suppress vestibular function in some patients with a local application of gentamicin without damaging auditory function. Variations in round window membrane permeability and in perilymph flow could account for why hearing losses are observed in some patients. PMID:17603318

  13. The properties of the outer membrane localized Lipid A transporter LptD

    NASA Astrophysics Data System (ADS)

    Haarmann, Raimund; Ibrahim, Mohamed; Stevanovic, Mara; Bredemeier, Rolf; Schleiff, Enrico

    2010-11-01

    Gram-negative bacteria are surrounded by a cell wall including the outer membrane. The outer membrane is composed of two distinct monolayers where the outer layer contains lipopolysaccharides (LPS) with the non-phospholipid Lipid A as the core. The synthesis of Lipid A is initiated in the cytosol and thereby the molecule has to be transported across the inner and outer membranes. The β-barrel lipopolysaccharide-assembly protein D (LptD) was discovered to be involved in the transfer of Lipid A into the outer membrane of Gram-negative bacteria. At present the molecular procedure of lipid transfer across the outer membrane remains unknown. Here we approached the functionality of the transfer system by an electrophysiological analysis of the outer membrane protein from Escherichia coli named ecLptD. In vitro the protein shows cation selectivity and has an estimated pore diameter of about 1.8 nm. Addition of Lipid A induces a transition of the open state to a sub-conductance state with two independent off-rates, which might suggest that LptD is able to bind and transport the molecule in vitro. To generalize our findings with respect to the Lipid A transport system of other Gram-negative bacteria we have explored the existence of the proteins involved in this pathway by bioinformatic means. We were able to identify the membrane-inserted components of the Lipid A transport system in all Gram-negative bacteria, whereas the periplasmic components appear to be species-specific. The LptD proteins of different bacteria are characterized by their periplasmic N-terminal domain and a C-terminal barrel region. The latter shows distinct sequence properties, particularly in LptD proteins of cyanobacteria, and this specific domain can be found in plant proteins as well. By electrophysiological experiments on LptD from Anabaena sp. PCC 7120 we are able to confirm the functional relation of anaLptD to Lipid A transport.

  14. Unmasking local activity within local field potentials (LFPs) by removing distal electrical signals using independent component analysis

    PubMed Central

    Whitmore, Nathan W.; Lin, Shih-Chieh

    2016-01-01

    Local field potentials (LFPs) are commonly thought to reflect the aggregate dynamics in local neural circuits around recording electrodes. However, we show that when LFPs are recorded in awake behaving animals against a distal reference on the skull as commonly practiced, LFPs are significantly contaminated by non-local and non-neural sources arising from the reference electrode and from movement-related noise. In a data set with simultaneously recorded LFPs and electroencephalograms (EEGs) across multiple brain regions while rats perform an auditory oddball task, we used independent component analysis (ICA) to identify signals arising from electrical reference and from volume-conducted noise based on their distributed spatial pattern across multiple electrodes and distinct power spectral features. These sources of distal electrical signals collectively accounted for 23–77% of total variance in unprocessed LFPs, as well as most of the gamma oscillation responses to the target stimulus in EEGs. Gamma oscillation power was concentrated in volume-conducted noise and was tightly coupled with the onset of licking behavior, suggesting a likely origin of muscle activity associated with body movement or orofacial movement. The removal of distal signal contamination also selectively reduced correlations of LFP/EEG signals between distant brain regions but not within the same region. Finally, the removal of contamination from distal electrical signals preserved an event-related potential (ERP) response to auditory stimuli in the frontal cortex and also increased the coupling between the frontal ERP amplitude and neuronal activity in the basal forebrain, supporting the conclusion that removing distal electrical signals unmasked local activity within LFPs. Together, these results highlight the significant contamination of LFPs by distal electrical signals and caution against the straightforward interpretation of unprocessed LFPs. Our results provide a principled approach to

  15. Local sparse component analysis for blind source separation: an application to resting state FMRI.

    PubMed

    Vieira, Gilson; Amaro, Edson; Baccala, Luiz A

    2014-01-01

    We propose a new Blind Source Separation technique for whole-brain activity estimation that best profits from FMRI's intrinsic spatial sparsity. The Local Sparse Component Analysis (LSCA) combines wavelet analysis, group-separable regularizers, contiguity-constrained clusterization and principal components analysis (PCA) into a unique spatial sparse representation of FMRI images towards efficient dimensionality reduction without sacrificing physiological characteristics by avoiding artificial stochastic model constraints. The LSCA outperforms classical PCA source reconstruction for artificial data sets over many noise levels. A real FMRI data illustration reveals resting-state activities in regions hard to observe, such as thalamus and basal ganglia, because of their small spatial scale.

  16. Adding local components to global functions for continuous covariates in multivariable regression modeling.

    PubMed

    Binder, H; Sauerbrei, W

    2010-03-30

    When global techniques, based on fractional polynomials (FPs), are employed for modeling potentially nonlinear effects of several continuous covariates on a response, accessible model equations are obtained. However, local features might be missed. Therefore, a procedure is introduced, which systematically checks model fits, obtained by the multivariable fractional polynomial (MFP) approach, for overlooked local features. Statistically significant local polynomials are then parsimoniously added. This approach, called MFP + L, is seen to result in an effective control of the Type I error with respect to the addition of local components in a small simulation study with univariate and multivariable settings. Prediction performance is compared with that of a penalized regression spline technique. In a setting unfavorable for FPs, the latter outperforms the MFP approach, if there is much information in the data. However, the addition of local features reduces this performance difference. There is only a small detrimental effect in settings where the MFP approach performs better. In an application example with children's respiratory health data, fits from the spline-based approach indicate many local features, but MFP + L adds only few significant features, which seem to have good support in the data. The proposed approach may be expected to be superior in settings with local features, but retains the good properties of the MFP approach in a large number of settings where global functions are sufficient.

  17. Localization of Drosophila retinal degeneration B, a membrane- associated phosphatidylinositol transfer protein

    PubMed Central

    1993-01-01

    The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160- kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium. PMID:8354691

  18. Lipid raft components cholesterol and sphingomyelin increase H+/OH− permeability of phosphatidylcholine membranes

    PubMed Central

    Gensure, Rebekah H.; Zeidel, Mark L.; Hill, Warren G.

    2006-01-01

    H+/OH− permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH− and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH− permeability from a baseline of 2.4×10−2 cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH− permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH− permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484–1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH− conductivity, (iii) H+/OH− fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH− permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results. PMID

  19. The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology.

    PubMed

    Lorenz, Holger; Hailey, Dale W; Wunder, Christian; Lippincott-Schwartz, Jennifer

    2006-01-01

    Correct localization and topology are crucial for the cellular function of a protein. To determine the topology of membrane proteins, a new technique, called the fluorescence protease protection (FPP) assay, can be applied. This assay uses the restricted proteolytic digestibility of GFP-tagged transmembrane proteins to indicate their intramembrane orientation. The sole requirements for FPP are the expression of GFP fusion proteins and the selective permeabilization of the plasma membrane, which permits a wide range of cell types and organelles to be investigated. The FPP assay can be carried out in a straightforward manner to obtain reliable results within minutes. Here we provide a step-by-step protocol for the assay. As an example, we use FPP to determine which terminus of an endoplasmic reticulum (ER) transmembrane protein is lumenal and which one is facing the cytosol.

  20. Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi.

    PubMed Central

    Brusca, J S; McDowall, A W; Norgard, M V; Radolf, J D

    1991-01-01

    Borrelia burgdorferi B31 with and without outer membranes contained nearly identical amounts of outer surface proteins A and B. The majority of each immunogen also was localized intracellularly by immunocryoultramicrotomy. These results are inconsistent with the widely held belief that outer surface proteins A and B are exclusively outer membrane proteins. Images FIG. 1 FIG. 2 FIG. 3 PMID:1744059

  1. The Saccharomyces cerevisiae spindle pole body (SPB) component Nbp1p is required for SPB membrane insertion and interacts with the integral membrane proteins Ndc1p and Mps2p.

    PubMed

    Araki, Yasuhiro; Lau, Corine K; Maekawa, Hiromi; Jaspersen, Sue L; Giddings, Thomas H; Schiebel, Elmar; Winey, Mark

    2006-04-01

    The spindle pole body (SPB) in Saccharomyces cerevisiae functions to nucleate and organize spindle microtubules, and it is embedded in the nuclear envelope throughout the yeast life cycle. However, the mechanism of membrane insertion of the SPB has not been elucidated. Ndc1p is an integral membrane protein that localizes to SPBs, and it is required for insertion of the SPB into the nuclear envelope during SPB duplication. To better understand the function of Ndc1p, we performed a dosage suppressor screen using the ndc1-39 temperature-sensitive allele. We identified an essential SPB component, Nbp1p. NBP1 shows genetic interactions with several SPB genes in addition to NDC1, and two-hybrid analysis revealed that Nbp1p binds to Ndc1p. Furthermore, Nbp1p is in the Mps2p-Bbp1p complex in the SPB. Immunoelectron microscopy confirmed that Nbp1p localizes to the SPB, suggesting a function at this location. Consistent with this hypothesis, nbp1-td (a degron allele) cells fail in SPB duplication upon depletion of Nbp1p. Importantly, these cells exhibit a "dead" SPB phenotype, similar to cells mutant in MPS2, NDC1, or BBP1. These results demonstrate that Nbp1p is a SPB component that acts in SPB duplication at the point of SPB insertion into the nuclear envelope.

  2. A membrane-proximal, C-terminal α-helix is required for plasma membrane localization and function of the G Protein-coupled receptor (GPCR) TGR5.

    PubMed

    Spomer, Lina; Gertzen, Christoph G W; Schmitz, Birte; Häussinger, Dieter; Gohlke, Holger; Keitel, Verena

    2014-02-07

    The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of β-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the β2 adrenergic receptor (β2AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5β2AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the β2AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.

  3. Multiple sound source localization using gammatone auditory filtering and direct sound componence detection

    NASA Astrophysics Data System (ADS)

    Chen, Huaiyu; Cao, Li

    2017-06-01

    In order to research multiple sound source localization with room reverberation and background noise, we analyze the shortcomings of traditional broadband MUSIC and ordinary auditory filtering based broadband MUSIC method, then a new broadband MUSIC algorithm with gammatone auditory filtering of frequency component selection control and detection of ascending segment of direct sound componence is proposed. The proposed algorithm controls frequency component within the interested frequency band in multichannel bandpass filter stage. Detecting the direct sound componence of the sound source for suppressing room reverberation interference is also proposed, whose merits are fast calculation and avoiding using more complex de-reverberation processing algorithm. Besides, the pseudo-spectrum of different frequency channels is weighted by their maximum amplitude for every speech frame. Through the simulation and real room reverberation environment experiments, the proposed method has good performance. Dynamic multiple sound source localization experimental results indicate that the average absolute error of azimuth estimated by the proposed algorithm is less and the histogram result has higher angle resolution.

  4. Slc26a7 chloride channel activity and localization in mouse Reissner's membrane epithelium.

    PubMed

    Kim, Kyunghee X; Sanneman, Joel D; Kim, Hyoung-Mi; Harbidge, Donald G; Xu, Jie; Soleimani, Manoocher; Wangemann, Philine; Marcus, Daniel C

    2014-01-01

    Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner's membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner's membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO3- over Cl- and inhibited by I- and NPPB. Elevated NO3- currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Cl- transport in Reissner's membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Cl- conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner's membrane cells during local perturbations of pH.

  5. Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2

    PubMed Central

    Barrales, Ramón Ramos; Forn, Marta; Georgescu, Paula Raluca; Sarkadi, Zsuzsa; Braun, Sigurd

    2016-01-01

    Transcriptionally silent chromatin localizes to the nuclear periphery, which provides a special microenvironment for gene repression. A variety of nuclear membrane proteins interact with repressed chromatin, yet the functional role of these interactions remains poorly understood. Here, we show that, in Schizosaccharomyces pombe, the nuclear membrane protein Lem2 associates with chromatin and mediates silencing and heterochromatin localization. Unexpectedly, we found that these functions can be separated and assigned to different structural domains within Lem2, excluding a simple tethering mechanism. Chromatin association and tethering of centromeres to the periphery are mediated by the N-terminal LEM (LAP2–Emerin–MAN1) domain of Lem2, whereas telomere anchoring and heterochromatin silencing require exclusively its conserved C-terminal MSC (MAN1–Src1 C-terminal) domain. Particularly, silencing by Lem2 is epistatic with the Snf2/HDAC (histone deacetylase) repressor complex SHREC at telomeres, while its necessity can be bypassed by deleting Epe1, a JmjC protein with anti-silencing activity. Furthermore, we found that loss of Lem2 reduces heterochromatin association of SHREC, which is accompanied by increased binding of Epe1. This reveals a critical function of Lem2 in coordinating these antagonistic factors at heterochromatin. The distinct silencing and localization functions mediated by Lem2 suggest that these conserved LEM-containing proteins go beyond simple tethering to play active roles in perinuclear silencing. PMID:26744419

  6. Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2.

    PubMed

    Barrales, Ramón Ramos; Forn, Marta; Georgescu, Paula Raluca; Sarkadi, Zsuzsa; Braun, Sigurd

    2016-01-15

    Transcriptionally silent chromatin localizes to the nuclear periphery, which provides a special microenvironment for gene repression. A variety of nuclear membrane proteins interact with repressed chromatin, yet the functional role of these interactions remains poorly understood. Here, we show that, in Schizosaccharomyces pombe, the nuclear membrane protein Lem2 associates with chromatin and mediates silencing and heterochromatin localization. Unexpectedly, we found that these functions can be separated and assigned to different structural domains within Lem2, excluding a simple tethering mechanism. Chromatin association and tethering of centromeres to the periphery are mediated by the N-terminal LEM (LAP2-Emerin-MAN1) domain of Lem2, whereas telomere anchoring and heterochromatin silencing require exclusively its conserved C-terminal MSC (MAN1-Src1 C-terminal) domain. Particularly, silencing by Lem2 is epistatic with the Snf2/HDAC (histone deacetylase) repressor complex SHREC at telomeres, while its necessity can be bypassed by deleting Epe1, a JmjC protein with anti-silencing activity. Furthermore, we found that loss of Lem2 reduces heterochromatin association of SHREC, which is accompanied by increased binding of Epe1. This reveals a critical function of Lem2 in coordinating these antagonistic factors at heterochromatin. The distinct silencing and localization functions mediated by Lem2 suggest that these conserved LEM-containing proteins go beyond simple tethering to play active roles in perinuclear silencing.

  7. Lateral organization and domain formation in a two-component lipid membrane system.

    PubMed Central

    Leidy, C; Wolkers, W F; Jørgensen, K; Mouritsen, O G; Crowe, J H

    2001-01-01

    The thermodynamic phase behavior and lateral lipid membrane organization of unilamellar vesicles made from mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) were investigated by fluorescence resonance energy transfer (FRET) as a function of temperature and composition. This was done by incorporating a headgroup-labeled lipid donor (NBD-DPPE) and acceptor (N-Rh-DPPE) in low concentrations into the binary mixtures. Two instances of increased energy transfer efficiency were observed close to the phase lines in the DMPC/DSPC phase diagram. The increase in energy transfer efficiency was attributed to a differential preference of the probes for dynamic and fluctuating gel/fluid coexisting phases. This differential preference causes the probes to segregate (S. Pedersen, K. Jørgensen, T. R. Baekmark, and O. G. Mouritsen, 1996, Biophys. J. 71:554-560). The observed increases in energy transfer match with the boundaries of the DMPC/DSPC phase diagram, as measured by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). We propose that the two instances of probe segregation are due to the presence of DMPC-rich and DSPC-rich domains, which form a dynamic structure of gel/fluid coexisting phases at two different temperatures. Monitoring the melting profile of each lipid component independently by FTIR shows that the domain structure is formed by DMPC-rich and DSPC-rich domains rather than by pure DMPC and DSPC domains. PMID:11259295

  8. Asymmetric Localization of Components and Regulators of the Mitotic Exit Network at Spindle Pole Bodies.

    PubMed

    Scarfone, Ilaria; Piatti, Simonetta

    2017-01-01

    Most proteins of the Mitotic Exit Network (MEN) and their upstream regulators localize at spindle pole bodies (SPBs) at least in some stages of the cell cycle. Studying the SPB localization of MEN factors has been extremely useful to elucidate their biological roles, organize them in a hierarchical pathway, and define their dynamics under different conditions.Recruitment to SPBs of the small GTPase Tem1 and the downstream kinases Cdc15 and Mob1/Dbf2 is thought to be essential for Cdc14 activation and mitotic exit, while that of the upstream Tem1 regulators (the Kin4 kinase and the GTPase activating protein Bub2-Bfa1) is important for MEN inhibition upon spindle mispositioning. Here, we describe the detailed fluorescence microscopy procedures that we use in our lab to analyze the localization at SPBs of Mitotic Exit Network (MEN) components tagged with GFP or HA epitopes.

  9. Poisoning of mixed matrix membranes by fermentation components in pervaporation of ethanol

    USDA-ARS?s Scientific Manuscript database

    Pervaporation is an alternative to distillation for recovering ethanol produced by fermentation of grains and biomass. Ethanol-selective mixed matrix membranes of the hydrophobic zeolite ZSM-5 in polydimethylsiloxane (PDMS) have superior performance compared to pure PDMS membranes in pervaporation o...

  10. Localization of the membrane attack complex (MAC) in experimental immune complex glomerulonephritis

    PubMed Central

    1983-01-01

    The role of the membrane attack complex (MAC) as a mediator of renal tissue injury was evaluated in rats affected by bovine serum albumin (BSA)-induced immune complex glomerulonephritis. Immunofluorescence studies revealed concurrent deposits of IgG, BSA, C3, and the MAC along glomerular capillary walls, although the MAC manifested a more restricted distribution than that observed for immune complexes. Immunoelectron microscopic techniques were utilized to demonstrate immune complexes, C3, and the MAC within dense deposits in the subepithelial aspect of the basement membrane. Visceral epithelial foot processes were fused in areas overlying large dense deposits and exhibited intense staining for the MAC, lesser reactivity for C3 but IgG was absent from the foot process membranes. Smaller granular deposits of immune complexes, C3, and the MAC were observed in the subendothelial region of the lamina rara interna and the lamina densa. Immune complexes may activate the classical complement pathway causing diffuse injury to the glomerular basement membrane (GBM), allowing subepithelial accumulation of complexes. These observations implicate the MAC as a mediator of GBM and juxtaposed podocyte membrane injury, thereby contributing to disruption of the glomerular filtration barrier. IgG and C3 were demonstrated within tubulointerstitial regions on the surface of collagen fibers in close proximity to the tubular basement membrane (TBM) of proximal convoluted tubules. Within the TBM, C3 localization was prominent with diminished reactivity for the MAC, but IgG was not detectable. The demonstration of C3 and scant MAC deposits in the TBM of nonimmunized control rats without evidence of interstitial IgG and C3 deposits suggests that both nonimmune and immune processes play a role in the pathogenesis of extraglomerular lesions. Evidence derived from these morphologic studies indicates that the MAC is associated with injury to the GBM, foot process membranes of visceral

  11. Cytological assessment of the epithelial cells of the nasal mucous membrane after local fluticasone therapy.

    PubMed

    Trybus, E; Krol, T; Obarzanowski, T; Trybus, W; Kopacz-Bednarska, A; Obarzanowski, M

    2015-02-01

    The majority of cytological studies concern the influence of glucocorticosteroids on cells involved in creating and sustaining inflammation, such as eosinophils or neutrophils. Much less attention is devoted to epithelial cells. It should also be noticed that glucocorticosteroid drugs administered nasally for local action can significantly change the cytological image of the nasal mucous membrane. Therefore, the purpose of this research was to cytologically assess the influence of topical fluticasone therapy on the nasal mucous membrane cells, with special attention for the changes in the morphology of epithelial cells. The research samples were taken from patients with symptoms of chronic rhinitis and suspected allergies. The research was a two-step process. In the first step, a smear was taken from the surface of the nasal mucous membrane of the above-mentioned patients before the start of therapy and the obtained cytological image was compared with a control image of the nasal mucous of healthy people. Step two involved the cytology of the same patients after 4 weeks of fluticasone therapy, applied as a nasal aerosol in two doses of 50 μg to each nostril once per day, in the combined daily dose of 200 μg (for adults and children aged 12 or more). Children aged between 4 and 12 were given a single dose of 50 μg to each nostril once per day, in a daily dose of 100 μg. Based on smears stained according to the Papanicolaou and Pappenheim method, a qualitative and quantitative analysis of changes in the mucous membrane of nasal cells was performed. The cytological assessment of nasal mucous membrane stains of patients with chronic rhinitis before fluticasone treatment enabled a diagnosis of chronic infectious rhinitis, compared through the presence of numerous neutrophils and bacteria. The studied samples did not show significant changes in the morphology of epithelial cells, only a few cells with mild vacuolation changes of the cytoplasm were found. The use of

  12. CAR-1, a protein that localizes with the mRNA decapping component DCAP-1, is required for cytokinesis and ER organization in Caenorhabditis elegans embryos.

    PubMed

    Squirrell, Jayne M; Eggers, Zachary T; Luedke, Nancy; Saari, Bonnie; Grimson, Andrew; Lyons, Gary E; Anderson, Philip; White, John G

    2006-01-01

    The division of one cell into two requires the coordination of multiple components. We describe a gene, car-1, whose product may provide a link between disparate cellular processes. Inhibition of car-1 expression in Caenorhabditis elegans embryos causes late cytokinesis failures: cleavage furrows ingress but subsequently regress and the spindle midzone fails to form, even though midzone components are present. The localized accumulation of membrane that normally develops at the apex of the cleavage furrow during the final phase of cytokinesis does not occur and organization of the endoplasmic reticulum is aberrant, indicative of a disruption in membrane trafficking. The car-1 gene has homologues in a number of species, including proteins that associate with RNA binding proteins. CAR-1 localizes to P-granules (germ-line specific ribonucleoprotein particles) and discrete, developmentally regulated cytoplasmic foci. These foci also contain DCAP-1, a protein involved in decapping mRNAs. Thus, CAR-1, a protein likely to be associated with RNA metabolism, plays an essential role in the late stage of cytokinesis, suggesting a novel link between RNA, membrane trafficking and cytokinesis in the C. elegans embryo.

  13. One-dimensional nonlinear elastodynamic models and their local conservation laws with applications to biological membranes.

    PubMed

    Cheviakov, A F; Ganghoffer, J-F

    2016-05-01

    The framework of incompressible nonlinear hyperelasticity and viscoelasticity is applied to the derivation of one-dimensional models of nonlinear wave propagation in fiber-reinforced elastic solids. Equivalence transformations are used to simplify the resulting wave equations and to reduce the number of parameters. Local conservation laws and global conserved quantities of the models are systematically computed and discussed, along with other related mathematical properties. Sample numerical solutions are presented. The models considered in the paper are appropriate for the mathematical description of certain aspects of the behavior of biological membranes and similar structures.

  14. Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

    PubMed Central

    Mendanha, S.A.; Anjos, J.L.V.; Silva, A.H.M.; Alonso, A.

    2012-01-01

    Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation. PMID:22473321

  15. Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium.

    PubMed

    Sun, Tong; Kim, Bohye; Kim, Lou W

    2013-10-01

    Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which the mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3(-) cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3(-) cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization.

  16. Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium

    PubMed Central

    Sun, Tong; Kim, Bohye; Kim, Lou W.

    2013-01-01

    Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3− cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3− cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization. PMID:24102085

  17. Localization of Components of the RNA-Degrading Machine in Bacillus subtilis

    PubMed Central

    Cascante-Estepa, Nora; Gunka, Katrin; Stülke, Jörg

    2016-01-01

    In bacteria, the control of mRNA stability is crucial to allow rapid adaptation to changing conditions. In most bacteria, RNA degradation is catalyzed by the RNA degradosome, a protein complex composed of endo- and exoribonucleases, RNA helicases, and accessory proteins. In the Gram-positive model organism Bacillus subtilis, the existence of a RNA degradosome assembled around the membrane-bound endoribonuclease RNase Y has been proposed. Here, we have studied the intracellular localization of the protein that have been implicated in the potential B. subtilis RNA degradosome, i.e., polynucleotide phosphorylase, the exoribonucleases J1 and J2, the DEAD-box RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase. Our data suggests that the bulk of these enzymes is located in the cytoplasm. The RNases J1 and J2 as well as the RNA helicase CshA were mainly localized in the peripheral regions of the cell where also the bulk of messenger RNA is localized. We were able to demonstrate active exclusion of these proteins from the transcribing nucleoid. Taken together, our findings suggest that the interactions of the enzymes involved in RNA degradation in B. subtilis are rather transient. PMID:27708634

  18. Local bisphosphonate reduces migration and formation of radiolucent lines adjacent to cemented acetabular components.

    PubMed

    Schilcher, J; Palm, L; Ivarsson, I; Aspenberg, P

    2017-03-01

    Post-operative migration of cemented acetabular components as measured by radiostereometric analysis (RSA) has a strong predictive power for late, aseptic loosening. Also, radiolucent lines predict late loosening. Migration has been reduced by systemic bisphosphonate treatment in randomised trials of hip and knee arthroplasty. Used as a local treatment, a higher local dose of bisphosphonate can be achieved without systemic exposure. We wished to see if this principle could be applied usefully in total hip arthroplasty (THA). In this randomised placebo-controlled, double-blinded trial with 60 participants, we compressed gauze soaked in bisphosphonate solution (ibandronate) or saline against the acetabular bone bed immediately before cementing the acetabular component. RSA, classification of radiolucent lines, the Harris Hip Score (HHS) and the Western Ontario McMasters Universities Osteoarthritis Index (WOMAC) were carried out at three-, six-, 12-, and 24-month follow-up. Migration of the cemented acetabular component relative to the pelvis was reduced by movement almost half in the ibandronate group, when measured as maximum total point or as movement of the femoral head (p = 0.001 and 0.004, respectively). Radiolucent lines after one year were classified as absent, partial or complete, and correlated with treatment (rho 0.37; p = 0.004). Only three of 30 patients in the ibandronate group had complete lines, compared with 13 of 28 in the placebo group (p = 0.002). There were no significant effects on HHS or WOMAC score. Considering the power of RSA to predict loosening of cemented acetabular components, and the likelihood that radiolucent lines indicate risk of loosening, these data suggest that local treatment with a bisphosphonate can reduce the risk of late aseptic loosening. Cite this article: Bone Joint J 2017;99-B:317-24. ©2017 The British Editorial Society of Bone & Joint Surgery.

  19. Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

    PubMed Central

    Andersen, J N; Elson, A; Lammers, R; Rømer, J; Clausen, J T; Møller, K B; Møller, N P

    2001-01-01

    To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function. PMID:11237862

  20. Crystal Structure of Escherichia coli CusC, the Outer Membrane Component of a Heavy Metal Efflux Pump

    PubMed Central

    Indic, Mridhu; van den Berg, Bert

    2011-01-01

    Background While copper has essential functions as an enzymatic co-factor, excess copper ions are toxic for cells, necessitating mechanisms for regulating its levels. The cusCBFA operon of E. coli encodes a four-component efflux pump dedicated to the extrusion of Cu(I) and Ag(I) ions. Methodology/Principal Findings We have solved the X-ray crystal structure of CusC, the outer membrane component of the Cus heavy metal efflux pump, to 2.3 Å resolution. The structure has the largest extracellular opening of any outer membrane factor (OMF) protein and suggests, for the first time, the presence of a tri-acylated N-terminal lipid anchor. Conclusions/Significance The CusC protein does not have any obvious features that would make it specific for metal ions, suggesting that the narrow substrate specificity of the pump is provided by other components of the pump, most likely by the inner membrane component CusA. PMID:21249122

  1. Effect of relative humidity cycles accompanied by intermittent start/stop switches on performance degradation of membrane electrode assembly components in proton exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Qiu, Yanling; Zhong, Hexiang; Wang, Meiri; Zhang, Huamin

    2015-06-01

    The performance degradation of membrane electrode assembly (MEA) components in proton exchange membrane fuel cell (PEMFC) is studied by designing relative humidity (RH) cycles accompanied by intermittent start/stop switches. Cathode catalyst activity, permeability and resistance of proton exchange membrane (PEM) as well as cell performance are monitored during the test procedure. The interfaces of MEA, the catalyst particle distribution near the cathode inlet are characterized by SEM and TEM, respectively. The results demonstrate both the overall H2 permeability and crossover current of PEM are doubled compared with its initial properties. Signs of PEM degradation, including periodical thinning, cracks and pinholes formation, are observed after 300 RH cycles and 40 times of start/stop switches. The average Pt particle size increases by more than 75%, and the cathode electrochemical surface area decreases by 48% after the test procedure. Meanwhile, the cathode catalyst layer becomes looser due to the dissolution of some smaller Pt particles and catalyst agglomeration in the RH cycles and the high potential during the intermittent start/stop switches. The membrane resistance demonstrates downshift variation during the RH cycles. PEMFC performance, however, decays due to the chemical and electrochemical attack as well as the mechanical stresses.

  2. Use of gene fusions to study outer membrane protein localization in Escherichia coli.

    PubMed Central

    Silhavy, T J; Shuman, H A; Beckwith, J; Schwartz, M

    1977-01-01

    Escherichia coli strains have been isolated that produce hybrid proteins comprised of an NH2-terminal sequence from the lamB gene product (an outer membrane protein) and a major portion of the COOH-terminal sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23; a cytoplasmic protein). These proteins exhibit beta-galactosidase activity. One such strain, pop 3105, produces a hybrid protein containing very little of the lamB gene protein; the protein is found in the cytoplasm. The protein found in a second strain, pop 3186, contains much more of the lamB gene protein; a substantial fraction of the beta-galactosidase activity is found in the outer membrane, probably facing outward. These results indicate that information necessary to direct the lamB gene product to its outer membrane location is located within the lamB gene itself. The properties of such fusion strains open up the prospect of a precise genetic analysis of the genetic components involved in protein transport. Images PMID:414221

  3. CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

    PubMed

    Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

    2000-06-01

    The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

  4. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    PubMed

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  5. RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels.

    PubMed

    Ogino, Kazutoyo; Low, Sean E; Yamada, Kenta; Saint-Amant, Louis; Zhou, Weibin; Muto, Akira; Asakawa, Kazuhide; Nakai, Junichi; Kawakami, Koichi; Kuwada, John Y; Hirata, Hiromi

    2015-03-03

    Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane.

  6. N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization

    PubMed Central

    De Napoli, MG; de Miguel, N; Lebrun, M; Moreno, SNJ; Angel, SO; Corvi, MM

    2013-01-01

    Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii. PMID:23485398

  7. Cooperative interactions of LPPR family members in membrane localization and alteration of cellular morphology

    PubMed Central

    Yu, Panpan; Agbaegbu, Chinyere; Malide, Daniela A.; Wu, Xufeng; Katagiri, Yasuhiro; Hammer, John A.; Geller, Herbert M.

    2015-01-01

    ABSTRACT The lipid phosphate phosphatase-related proteins (LPPRs), also known as plasticity-related genes (PRGs), are classified as a new brain-enriched subclass of the lipid phosphate phosphatase (LPP) superfamily. They induce membrane protrusions, neurite outgrowth or dendritic spine formation in cell lines and primary neurons. However, the exact roles of LPPRs and the mechanisms underlying their effects are not certain. Here, we present the results of a large-scale proteome analysis to determine LPPR1-interacting proteins using co-immunoprecipitation coupled to mass spectrometry. We identified putative LPPR1-binding proteins involved in various biological processes. Most interestingly, we identified the interaction of LPPR1 with its family member LPPR3, LPPR4 and LPPR5. Their interactions were characterized by co-immunoprecipitation and colocalization analysis using confocal and super-resolution microscopy. Moreover, co-expressing two LPPR members mutually elevated their protein levels, facilitated their plasma membrane localization and resulted in an increased induction of membrane protrusions as well as the phosphorylation of S6 ribosomal protein. Taken together, we revealed a new functional cooperation between LPPR family members and discovered for the first time that LPPRs likely exert their function through forming complex with its family members. PMID:26183180

  8. Fluorescence imaging of local membrane electric fields during the excitation of single neurons in culture.

    PubMed Central

    Gogan, P; Schmiedel-Jakob, I; Chitti, Y; Tyc-Dumont, S

    1995-01-01

    The spatial distribution of depolarized patches of membrane during the excitation of single neurons in culture has been recorded with a high spatial resolution (1 micron2/pixel) imaging system based on a liquid-nitrogen-cooled astronomical camera mounted on an inverted microscope. Images were captured from rat nodose neurons stained with the voltage-sensitive dye RH237. Conventional intracellular microelectrode recordings were made in synchrony with the images. During an action potential the fluorescence changes occurred in localized, unevenly distributed membrane areas, which formed clusters of depolarized sites of different sizes and intensities. When fast conductances were blocked by the addition of tetrodotoxin, a reduction in the number and the intensities of the depolarized sites was observed. The blockade by tetrodotoxin of voltage-clamped neurons also reduced the number of depolarized sites, although the same depolarizing voltage step was applied. Similarly, when a voltage-clamped neuron was depolarized by a constant-amplitude voltage step, the number of depolarized sites varied according to the degree of activation of the voltage-sensitive channels, which was modified by changing the holding potential. These results suggest that the spatial patterns of depolarization observed during excitation are related to the operations of ionic channels in the membrane. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:8527643

  9. Characterisation of the membrane-extrinsic domain of the TatB component of the twin arginine protein translocase.

    PubMed

    Maldonado, Barbara; Kneuper, Holger; Buchanan, Grant; Hatzixanthis, Kostas; Sargent, Frank; Berks, Ben C; Palmer, Tracy

    2011-02-04

    The twin arginine protein transport (Tat) system transports folded proteins across cytoplasmic membranes of bacteria and thylakoid membranes of plants, and in Escherichia coli it comprises TatA, TatB and TatC components. In this study we show that the membrane extrinsic domain of TatB forms parallel contacts with at least one other TatB protein. Truncation of the C-terminal two thirds of TatB still allows complex formation with TatC, although protein transport is severely compromised. We were unable to isolate transport-inactive single codon substitution mutations in tatB suggesting that the precise amino acid sequence of TatB is not critical to its function. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component, Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

    PubMed Central

    Šubrtová, Karolína; Panicucci, Brian; Zíková, Alena

    2015-01-01

    In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψm) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells. PMID:25714685

  11. Independent component analysis of EEG dipole source localization in resting and action state of brain

    NASA Astrophysics Data System (ADS)

    Almurshedi, Ahmed; Ismail, Abd Khamim

    2015-04-01

    EEG source localization was studied in order to determine the location of the brain sources that are responsible for the measured potentials at the scalp electrodes using EEGLAB with Independent Component Analysis (ICA) algorithm. Neuron source locations are responsible in generating current dipoles in different states of brain through the measured potentials. The current dipole sources localization are measured by fitting an equivalent current dipole model using a non-linear optimization technique with the implementation of standardized boundary element head model. To fit dipole models to ICA components in an EEGLAB dataset, ICA decomposition is performed and appropriate components to be fitted are selected. The topographical scalp distributions of delta, theta, alpha, and beta power spectrum and cross coherence of EEG signals are observed. In close eyes condition it shows that during resting and action states of brain, alpha band was activated from occipital (O1, O2) and partial (P3, P4) area. Therefore, parieto-occipital area of brain are active in both resting and action state of brain. However cross coherence tells that there is more coherence between right and left hemisphere in action state of brain than that in the resting state. The preliminary result indicates that these potentials arise from the same generators in the brain.

  12. Embryonal cell surface recognition. Extraction of an active plasma membrane component.

    PubMed

    Merrell, R; Gottlieb, D I; Glaser, L

    1975-07-25

    Plasma membranes obtained from different neural regions of the chicken embryo have previously been shown to specifically bind to homotypic cells and prevent cell aggregation (Merrell, R., and Glaser, L. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 2794-2798). Proteins responsible for the specific inhibition of cell aggregation have been solubilized from the plasma membrane of neural retina and optic tectum by delipidation with acetone followed by extraction with lithium diiodosalicylate. The extracts show the same regional and temporal specificity as previously shown for plasma membrane recognition by the same cells (Gottlieb, D. I., Merrell, R., and Glaser, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1800-1802). Two micrograms of the most purified protein fraction inhibits the aggregation of 2.5 times 10(-4) cells under standard assay conditions. This represents a 20-fold increase in specific activity compared to whole membranes.

  13. Perkinsus marinus superoxide dismutase 2 (PmSOD2) localizes to single-membrane subcellular compartments

    SciTech Connect

    Fernandez-Robledo, Jose A.; Schott, Eric J.; Vasta, Gerardo R.

    2008-10-17

    Perkinsus marinus (Phylum Perkinsozoa), a protozoan parasite of oysters, is considered one of the earliest diverging groups of the lineage leading to dinoflagellates. Perkinsus trophozoites are phagocytosed by oyster hemocytes, where they are likely exposed to reactive oxygen species. As part of its reactive oxygen detoxifying pathway, P. marinus possesses two iron-cofactored SOD (PmSOD1 and PmSOD2). Immunoflourescence analysis of P. marinus trophozoites and gene complementation in yeast revealed that PmSOD1 is targeted to the mitochondria. Surprisingly, although PmSOD2 is characterized by a bipartite N-terminus extension typical of plastid targeting, in preliminary immunofluorescence studies it was visualized as punctuate regions in the cytoplasm that could not be assigned to any organelle. Here, we used immunogold electron microscopy to examine the subcellular localization PmSOD2 in P. marinus trophozoites. Gold grains were mostly associated with single-membrane vesicle-like structures, and eventually, localized to electron-dense, apparently amorphous material present in the lumen of a larger, unique compartment. The images suggested that PmSOD2 is targeted to small vesicles that fuse and/or discharge their content into a larger compartment, possibly the large vacuole typical of the mature trophozoites. In light of the in silico targeting prediction, the association of PmSOD2 with single-membrane compartments raises interesting questions regarding its organellar targeting, and the nature of a putative relic plastid in Perkinsus species.

  14. Localization of connexin 30 in the luminal membrane of cells in the distal nephron.

    PubMed

    McCulloch, Fiona; Chambrey, Régine; Eladari, Dominique; Peti-Peterdi, János

    2005-12-01

    Several isoforms of the gap junction protein connexin (Cx) have been identified in a variety of tissues that communicate intercellular signals between adjacent cells. In the kidney, Cx37, Cx40, and Cx43 are localized in the vasculature, glomerulus, and tubular segments in a punctuate pattern, typical of classic gap junction channels. We performed immunohistochemistry in the mouse, rat, and rabbit kidney to study the localization of Cx30 protein, a new member of the Cx family. The vasculature, glomerulus, and proximal nephron segments were devoid of staining in all three species. Unexpectedly, Cx30 was found throughout the luminal membrane of select cells in the distal nephron. Expression of Cx30 was highest in the rat, which also showed some diffuse cytosolic labeling, continuous from the medullary thick ascending limb to the collecting duct system, and with the highest level in the distal convoluted tubule. Labeling in the mouse and rabbit was much less, limited to intercalated cells in the connecting segment and cortical collecting duct, where the apical signal was particularly strong. A high-salt-containing diet and culture medium upregulated Cx30 expression in the rat inner medulla and in M1 cells, respectively. The distinct, continuous labeling of the luminal plasma membrane and upregulation by high salt suggest that Cx30 may function as a hemichannel involved in the regulation of salt reabsorption in the distal nephron.

  15. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

    PubMed Central

    Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

  16. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  17. EGFR controls IQGAP basolateral membrane localization and mitotic spindle orientation during epithelial morphogenesis

    PubMed Central

    Bañón-Rodríguez, Inmaculada; Gálvez-Santisteban, Manuel; Vergarajauregui, Silvia; Bosch, Minerva; Borreguero-Pascual, Arantxa; Martín-Belmonte, Fernando

    2014-01-01

    Establishing the correct orientation of the mitotic spindle is an essential step in epithelial cell division in order to ensure that epithelial tubules form correctly during organ development and regeneration. While recent findings have identified some of the molecular mechanisms that underlie spindle orientation, many aspects of this process remain poorly understood. Here, we have used the 3D-MDCK model system to demonstrate a key role for a newly identified protein complex formed by IQGAP1 and the epithelial growth factor receptor (EGFR) in controlling the orientation of the mitotic spindle. IQGAP1 is a scaffolding protein that regulates many cellular pathways, from cell-cell adhesion to microtubule organization, and its localization in the basolateral membrane ensures correct spindle orientation. Through its IQ motifs, IQGAP1 binds to EGFR, which is responsible for maintaining IQGAP1 in the basolateral membrane domain. Silencing IQGAP1, or disrupting the basolateral localization of either IQGAP1 or EGFR, results in a non-polarized distribution of NuMA, mitotic spindle misorientation and defects in single lumen formation. PMID:24421325

  18. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  19. IKAP localizes to membrane ruffles with filamin A and regulates actin cytoskeleton organization and cell migration.

    PubMed

    Johansen, Lars Dan; Naumanen, Tiina; Knudsen, Astrid; Westerlund, Nina; Gromova, Irina; Junttila, Melissa; Nielsen, Christina; Bøttzauw, Trine; Tolkovsky, Aviva; Westermarck, Jukka; Coffey, Eleanor T; Jäättelä, Marja; Kallunki, Tuula

    2008-03-15

    Loss-of-function mutations in the IKBKAP gene, which encodes IKAP (ELP1), cause familial dysautonomia (FD), with defective neuronal development and maintenance. Molecular mechanisms leading to FD are poorly understood. We demonstrate that various RNA-interference-based depletions of IKAP lead to defective adhesion and migration in several cell types, including rat primary neurons. The defects could be rescued by reintroduction of wild-type IKAP but not by FD-IKAP, a truncated form of IKAP constructed according to the mutation found in the majority of FD patients. Cytosolic IKAP co-purified with proteins involved in cell migration, including filamin A, which is also involved in neuronal migration. Immunostaining of IKAP and filamin A revealed a distinct co-localization of these two proteins in membrane ruffles. Depletion of IKAP resulted in a significant decrease in filamin A localization in membrane ruffles and defective actin cytoskeleton organization, which both could be rescued by the expression of wild-type IKAP but not by FD-IKAP. No downregulation in the protein levels of paxillin or beclin 1, which were recently described as specific transcriptional targets of IKAP, was detected. These results provide evidence for the role of the cytosolic interactions of IKAP in cell adhesion and migration, and support the notion that cell-motility deficiencies could contribute to FD.

  20. Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

    DOE PAGES

    Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; ...

    2016-05-27

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed betweenmore » the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.« less

  1. Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

    SciTech Connect

    Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; Lee, Sang -Wook; Sohn, Youngjoo; Oh, Sang -Hyun; Parikh, Atul N.; Lee, Sin -Doo

    2016-05-27

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.

  2. Sensing small molecule interactions with lipid membranes by local pH modulation.

    PubMed

    Huang, Da; Zhao, Tao; Xu, Wei; Yang, Tinglu; Cremer, Paul S

    2013-11-05

    Herein, we utilized a label-free sensing platform based on pH modulation to detect the interactions between tetracaine, a positively charged small molecule used as a local anesthetic, and planar supported lipid bilayers (SLBs). The SLBs were patterned inside a flow cell, allowing for various concentrations of tetracaine to be introduced over the surface in a buffer solution. Studies with membranes containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) yielded an equilibrium dissociation constant value of Kd = 180 ± 47 μm for this small molecule-membrane interaction. Adding cholesterol to the SLBs decreased the affinity between tetracaine and the bilayers, while this interaction tightened when POPE (1-hexadecanoyl-2-(9-Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) was added. Studies were also conducted with three negatively charged membrane lipids, POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt)), and ganglioside GM1. All three measurements gave rise to a similar tightening of the apparent Kd value compared with pure POPC membranes. The lack of chemical specificity with the identity of the negatively charged lipid indicated that the tightening was largely electrostatic. Through a direct comparison with ITC measurements, it was found that the pH modulation sensor platform offers a facile, inexpensive, highly sensitive, and rapid method for the detection of interactions between putative drug candidates and lipid bilayers. As such, this technique may potentially be exploited as a screen for drug development and analysis.

  3. Progesterone receptor membrane component 1 deficiency attenuates growth while promoting chemosensitivity of human endometrial xenograft tumors

    PubMed Central

    Friel, Anne M.; Zhang, Ling; Pru, Cindy A.; Clark, Nicole C.; McCallum, Melissa L.; Blok, Leen J.; Shioda, Toshi; Peluso, John J.; Rueda, Bo R.; Pru, James K.

    2014-01-01

    Endometrial cancer is the leading gynecologic cancer in women in the United States with 52,630 women predicted to be diagnosed with the disease in 2014. The objective of this study was to determine if progesterone (P4) receptor membrane component 1 (PGRMC1) influenced endometrial cancer cell viability in response to chemotherapy in vitro and in vivo. A Jentiviral-based shRNA knockdown approach was used to generate stable PGRMC1-intact and PGRMC1-deplete Ishikawa endometrial cancer cell lines that also lacked expression of the classical progesterone receptor (PGR). Progesterone treatment inhibited mitosis of PGRMC1-intact, but not PGRMC1-deplete cells, suggesting that PGRMC1 mediates the anti-mitotic actions of P4.To test the hypothesis that PGRMC1 attenuates chemotherapy-induced apoptosis, PGRMC1-intact and PGRMC1-deplete cells were treated in vitro with vehicle, P4 (1 μM), doxorubicin (Dox. 2 μg/ml). or P4 + Dox for 48 h. Doxorubicin treatment of PGRMC1-intact cells resulted in a significant increase in cell death; however, co-treatment with P4 significantly attenuated Dex-induced cell death. This response to P4 was lost in PGRMC1-deplete cells. To extend these observations in vivo, a xenograft model was employed where PGRMC1-intact and PGRMC1-deplete endometrial tumors were generated following subcutaneous and intraperitonea l inoculation of immunocompromised NOD/SCIO and nude mice, respectively. Tumors derived from PGRMC1-deplete cells grew slower than tumors from PGRMC1-intact cells. Mice harboring endometrial tumors were then given three treatments of vehicle (1:1 cremophor EL: ethanol + 0.9% saline) or chemotherapy [Paclitaxel (15 mg/kg, i.p.) followed after an interval of 30 minutes by CARBOplatin (SO mg/kg)] at five day intervals. In response to chemotherapy, tumor volume decreased approximately four-fold more in PGRMC1-deplete tumors when compared with PGRMC1 intact control tumors, suggesting that PGRMC1 promotes tumor cell viability during

  4. Progesterone receptor membrane component 1 (PGRMC1) expression in murine retina

    PubMed Central

    Shanmugam, Arul K.; Mysona, Barbara A.; Wang, Jing; Zhao, Jing; Tawfik, Amany; Sanders, A.; Markand, Shanu; Zorrilla, Eric; Ganapathy, Vadivel; Bollinger, Kathryn E.; Smith, Sylvia B.

    2015-01-01

    Purpose Sigma receptor 1 (σR1) and 2 (σR2) are thought to be two distinct proteins which share the ability to bind multiple ligands, several of which are common to both receptors. Whether σR1 and σR2 share overlapping biological functions is unknown. Recently, progesterone receptor membrane component 1 (PGRMC1) was shown to contain the putative σR2 binding site. PGRMC1 has not been studied in retina. We hypothesize that biological interactions between σR1 and PGRMC1 will be evidenced by compensatory upregulation of PGRMC1 in σR1−/− mice. Methods Immunofluorescence, RT-PCR, and immunoblotting methods were used to analyze expression of PGRMC1 in wild type mouse retina. Tissues from σR1−/− mice were used to investigate whether a biological interaction exists between σR1 and PGRMC1. Results In the eye, PGRMC1 is expressed in corneal epithelium, lens, ciliary body epithelium, and retina. In retina, PGRMC1 is present in Müller cells and retinal pigment epithelium. This expression pattern is similar, but not identical to σR1. PGRMC1 protein levels in neural retina and eye cup from σR1−/− mice did not differ from wild type mice. Nonocular tissues, lung, heart, and kidney showed similar Pgrmc1 gene expression in wild type and σR1−/− mice. In contrast, liver, brain and intestine showed increased Pgrmc1 gene expression in σR1−/− mice. Conclusion Despite potential biological overlap, deletion of σR1 did not result in a compensatory change in PGRMC1 protein levels in σR1−/− mouse retina. Increased Pgrmc1 gene expression in organs with high lipid content such as liver, brain, and intestine indicate a possible tissue specific interaction between σR1 and PGRMC1. The current studies establish the presence of PGRMC1 in retina and lay the foundation for analysis of its biological function. PMID:26642738

  5. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

    PubMed

    Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  6. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane

    PubMed Central

    Richardson, Lynn G. L.; Paila, Yamuna D.; Siman, Steven R.; Chen, Yi; Smith, Matthew D.; Schnell, Danny J.

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus. PMID:24966864

  7. A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

    PubMed Central

    Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

    2016-01-01

    An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ∼10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures. PMID:27708275

  8. Species-specificity of the BamA component of the bacterial outer membrane protein-assembly machinery.

    PubMed

    Volokhina, Elena B; Grijpstra, Jan; Beckers, Frank; Lindh, Erika; Robert, Viviane; Tommassen, Jan; Bos, Martine P

    2013-01-01

    The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP) in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA.

  9. Reliable characteristics and stabilization of on-membrane SOI MOSFET-based components heated up to 335 °C

    NASA Astrophysics Data System (ADS)

    Amor, S.; André, N.; Gérard, P.; Ali, S. Z.; Udrea, F.; Tounsi, F.; Mezghani, B.; Francis, L. A.; Flandre, D.

    2017-01-01

    In this work we investigate the characteristics and critical operating temperatures of on-membrane embedded MOSFETs from an experimental and analytical point of view. This study permits us to conclude the possibility of integrating electronic circuitry in the close vicinity of micro-heaters and hot operation transducers. A series of calibrations and measurements has been performed to examine the behaviors of transistors, inverters and diodes, actuated at high temperature, on a membrane equipped with an on-chip integrated micro-heater. The studied n- and p-channel body-tied partially-depleted MOSFETs and CMOS inverter are embedded in a 5 μm-thick membrane fabricated by back-side MEMS micromachining using SOI technology. It has been noted that a pre-stabilization step after the harsh post-CMOS processing, through an in situ high-temperature annealing using the micro-heater, is mandatory in order to stabilize the MOSFETs characteristics. The electrical characteristics and performance of the on-membrane MOS components are discussed when heated up to 335 °C. This study supports the possibility of extending the potential of the micro-hotplate concept, under certain conditions, by embedding more electronic functionalities on the interface of on-membrane-based sensors leading to better sensing and actuation performances and a total area reduction, particularly for environmental or industrial applications.

  10. A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

    NASA Astrophysics Data System (ADS)

    Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

    2016-10-01

    An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ~10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

  11. [The effect of N-bromoacetamide on the components of sodium channel inactivation in the membrane of Ranvier's node].

    PubMed

    Lonskiĭ, A V; Krutetskaia, Z I; Roshchina, N G; P'iankova, S V

    1991-05-01

    In voltage clamp experiments on the frog Ranvier node, the specific protein reagent, N-bromacetamide, significantly decelerates the sodium inactivation kinetics and makes it incomplete. After treatment with N-bromacetamide, both fast and slow inactivation time constants are increased and the proportion of inactivation components is changed favouring the slowly inactivating one in the wide range of membrane potentials. The results are consistent with a single channel population following the 3-state model of inactivation.

  12. Facilitating in vivo tumor localization by principal component analysis based on dynamic fluorescence molecular imaging.

    PubMed

    Gao, Yang; Chen, Maomao; Wu, Junyu; Zhou, Yuan; Cai, Chuangjian; Wang, Daliang; Luo, Jianwen

    2017-09-01

    Fluorescence molecular imaging has been used to target tumors in mice with xenograft tumors. However, tumor imaging is largely distorted by the aggregation of fluorescent probes in the liver. A principal component analysis (PCA)-based strategy was applied on the in vivo dynamic fluorescence imaging results of three mice with xenograft tumors to facilitate tumor imaging, with the help of a tumor-specific fluorescent probe. Tumor-relevant features were extracted from the original images by PCA and represented by the principal component (PC) maps. The second principal component (PC2) map represented the tumor-related features, and the first principal component (PC1) map retained the original pharmacokinetic profiles, especially of the liver. The distribution patterns of the PC2 map of the tumor-bearing mice were in good agreement with the actual tumor location. The tumor-to-liver ratio and contrast-to-noise ratio were significantly higher on the PC2 map than on the original images, thus distinguishing the tumor from its nearby fluorescence noise of liver. The results suggest that the PC2 map could serve as a bioimaging marker to facilitate in vivo tumor localization, and dynamic fluorescence molecular imaging with PCA could be a valuable tool for future studies of in vivo tumor metabolism and progression. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  13. Standard plane localization in ultrasound by radial component model and selective search.

    PubMed

    Ni, Dong; Yang, Xin; Chen, Xin; Chin, Chien-Ting; Chen, Siping; Heng, Pheng Ann; Li, Shengli; Qin, Jing; Wang, Tianfu

    2014-11-01

    Acquisition of the standard plane is crucial for medical ultrasound diagnosis. However, this process requires substantial experience and a thorough knowledge of human anatomy. Therefore it is very challenging for novices and even time consuming for experienced examiners. We proposed a hierarchical, supervised learning framework for automatically detecting the standard plane from consecutive 2-D ultrasound images. We tested this technique by developing a system that localizes the fetal abdominal standard plane from ultrasound video by detecting three key anatomical structures: the stomach bubble, umbilical vein and spine. We first proposed a novel radial component-based model to describe the geometric constraints of these key anatomical structures. We then introduced a novel selective search method which exploits the vessel probability algorithm to produce probable locations for the spine and umbilical vein. Next, using component classifiers trained by random forests, we detected the key anatomical structures at their probable locations within the regions constrained by the radial component-based model. Finally, a second-level classifier combined the results from the component detection to identify an ultrasound image as either a "fetal abdominal standard plane" or a "non- fetal abdominal standard plane." Experimental results on 223 fetal abdomen videos showed that the detection accuracy of our method was as high as 85.6% and significantly outperformed both the full abdomen and the separate anatomy detection methods without geometric constraints. The experimental results demonstrated that our system shows great promise for application to clinical practice.

  14. Damage localization in linear-form structures based on sensitivity investigation for principal component analysis

    NASA Astrophysics Data System (ADS)

    Viet Ha, Nguyen; Golinval, Jean-Claude

    2010-10-01

    This paper addresses the problem of damage detection and localization in linear-form structures. Principal component analysis (PCA) is a popular technique for dynamic system investigation. The aim of the paper is to present a damage diagnosis method based on sensitivities of PCA results in the frequency domain. Starting from frequency response functions (FRFs) measured at different locations on the structure; PCA is performed to determine the main features of the signals. Sensitivities of principal directions obtained from PCA to structural parameters are then computed and inspected according to the location of sensors; their variation from the healthy state to the damaged state indicates damage locations. It is worth noting that damage localization is performed without the need of modal identification. Influences of some features as noise, choice of parameter and number of sensors are discussed. The efficiency and limitations of the proposed method are illustrated using numerical and real-world examples.

  15. The relative contributions of global and local acceleration components on speed perception and discriminability following adaptation.

    PubMed

    Hietanen, Markus A

    2015-10-01

    The perception of speed is dependent on the history of previously presented speeds. Adaptation to a given speed regularly results in a reduction of perceived speed and an increase in speed discriminability and in certain circumstances can result in an increase in perceived speed. In order to determine the relative contributions of the local and global speed components on perceived speed, this experiment used expanding dot flow fields with accelerating (global), decelerating (global) and mixed accelerating/decelerating (local) speed patterns. Profound decreases in perceived speed are found when viewing low test speeds after adaptation to high speeds. Small increases in the perceived speed of high test speeds occur following adaptation to low speeds. There were small but significant differences in perceived stimulus speed after adaptation due to different acceleration profiles. No evidence for global modulation of speed discriminability following adaptation was found. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Intracellular localization of membrane-bound ATPases in the compartmentalized anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’

    PubMed Central

    van Niftrik, Laura; van Helden, Mary; Kirchen, Silke; van Donselaar, Elly G; Harhangi, Harry R; Webb, Richard I; Fuerst, John A; Op den Camp, Huub J M; Jetten, Mike S M; Strous, Marc

    2010-01-01

    Anaerobic ammonium-oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out- to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by membrane-bound ATPases. To test this hypothesis, we investigated the location of membrane-bound ATPases in the anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F-ATPase, two atypical F-ATPases and a prokaryotic V-ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F-ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane-bounded compartment with a specific cellular function: energy metabolism. PMID:20545867

  17. [Characterization of peripheral erythron component in patients with localized thermal burns].

    PubMed

    Riazantseva, N V; Novitskiĭ, V V; Riazantsev, V P

    2000-06-01

    Cytospectrophotometry and electron microscopy of peripheral blood erythrocytes in patients with local surface thermal burns and healthy volunteers showed a notable decrease in the levels of sulfhydryl groups and lipoproteins, decreased number of biconcave discocytes, and increased number of transitional, prehemolytical, and degenerative forms of red blood cells. These changes were more pronounced during the early posttraumatic period and persisted for 2 months after burns were inflicted. Deep structural metabolic and morphological disorganization of the erythron peripheral component is a factor of risk of postburn complications.

  18. Defining a local arterial input function for perfusion MRI using independent component analysis.

    PubMed

    Calamante, Fernando; Mørup, Morten; Hansen, Lars Kai

    2004-10-01

    Quantification of cerebral blood flow (CBF) using dynamic-susceptibility contrast MRI relies on the deconvolution of the arterial input function (AIF), which is commonly estimated from the signal changes in a major artery. However, it has been shown that the presence of bolus delay/dispersion between the artery and the tissue of interest can be a significant source of error. These effects could be minimized if a local AIF were used, although the measurement of a local AIF can be problematic. This work describes a new methodology to define a local AIF using independent component analysis (ICA). The methodology was tested on data from patients with various cerebrovascular abnormalities and compared to the conventional approach of using a global AIF. The new methodology produced higher CBF and shorter mean transit time values (compared to the global AIF case) in areas with distorted AIFs, suggesting that the effects of delay/dispersion are minimized. The minimization of these effects using the calculated local AIF should lead to a more accurate quantification of CBF, which can have important implications for diagnosis and management of patients with cerebral ischemia.

  19. Components of RNA granules affect their localization and dynamics in neuronal dendrites.

    PubMed

    Mitsumori, Kazuhiko; Takei, Yosuke; Hirokawa, Nobutaka

    2017-04-12

    In neurons, RNA transport is important for local protein synthesis. Messenger RNAs (mRNAs) are transported along dendrites as large RNA granules. The localization and dynamics of Puralpha and Stau1, major components of RNA transport granules, were investigated in cultured hippocampal neurons. Puralpha-positive granules were localized in both the shafts and spines of dendrites. In contrast, Stau1-positive granules tended to be localized mainly in dendritic shafts. More than 90% of Puralpha-positive granules were positive for Stau1 in immature dendrites, while only half were positive in mature dendrites. Stau1-negative Puralpha granules tended to be stationary with fewer anterograde and retrograde movements than Stau1-positive Puralpha granules. After metabotropic glutamate receptor 5 (mGluR5) activation, Stau-1 positive granules remained in the dendritic shafts, while Puralpha granules translocated from the shaft to the spine. The translocation of Puralpha granules was dependent on Myosin Va, an actin-based molecular motor protein. Collectively, our findings suggest the possibility that the loss of Stau1 in Puralpha-positive RNA granules might promote their activity-dependent translocation into dendritic spines, which could underlie the regulation of protein synthesis in synapses.

  20. Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation.

    PubMed Central

    Mustardy, L; Los, D A; Gombos, Z; Murata, N

    1996-01-01

    There are four acyl-lipid desaturases in the cyanobacterium Synechocystis sp. PCC 6803. Each of these desaturases introduces a double bond at a specific position, such as the Delta6, Delta9, Delta12, or omicron3 position, in C18 fatty acids. The localization of the desaturases in cyanobacterial cells was examined immunocytochemically with antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-terminal regions of the desaturases. All four desaturases appeared to be located in the regions of both the cytoplasmic and the thylakoid membranes. These findings suggest that fatty acid desaturation of membrane lipids takes place in the thylakoid membranes as well as in the cytoplasmic membranes. Images Fig. 1 Fig. 2 Fig. 3 PMID:11607709

  1. Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

    PubMed

    Bedbrook, Claire N; Kato, Mihoko; Ravindra Kumar, Sripriya; Lakshmanan, Anupama; Nath, Ravi D; Sun, Fei; Sternberg, Paul W; Arnold, Frances H; Gradinaru, Viviana

    2015-08-20

    Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Plasma Membrane Localization Is Required for RasA-Mediated Polarized Morphogenesis and Virulence of Aspergillus fumigatus

    PubMed Central

    Juvvadi, Praveen R.; Rogg, Luise E.; Asfaw, Yohannes G.; Burns, Kimberlie A.; Randell, Scott H.; Steinbach, William J.

    2012-01-01

    Ras is a highly conserved GTPase protein that is essential for proper polarized morphogenesis of filamentous fungi. Localization of Ras proteins to the plasma membrane and endomembranes through posttranslational addition of farnesyl and palmitoyl residues is an important mechanism through which cells provide specificity to Ras signal output. Although the Aspergillus fumigatus RasA protein is known to be a major regulator of growth and development, the membrane distribution of RasA during polarized morphogenesis and the role of properly localized Ras signaling in virulence of a pathogenic mold remain unknown. Here we demonstrate that Aspergillus fumigatus RasA localizes primarily to the plasma membrane of actively growing hyphae. We show that treatment with the palmitoylation inhibitor 2-bromopalmitate disrupts normal RasA plasma membrane association and decreases hyphal growth. Targeted mutations of the highly conserved RasA palmitoylation motif also mislocalized RasA from the plasma membrane and led to severe hyphal abnormalities, cell wall structural changes, and reduced virulence in murine invasive aspergillosis. Finally, we provide evidence that proper RasA localization is independent of the Ras palmitoyltransferase homolog, encoded by erfB, but requires the palmitoyltransferase complex subunit, encoded by erfD. Our results demonstrate that plasma membrane-associated RasA is critical for polarized morphogenesis, cell wall stability, and virulence in A. fumigatus. PMID:22562470

  3. Plasma membrane localization is required for RasA-mediated polarized morphogenesis and virulence of Aspergillus fumigatus.

    PubMed

    Fortwendel, Jarrod R; Juvvadi, Praveen R; Rogg, Luise E; Asfaw, Yohannes G; Burns, Kimberlie A; Randell, Scott H; Steinbach, William J

    2012-08-01

    Ras is a highly conserved GTPase protein that is essential for proper polarized morphogenesis of filamentous fungi. Localization of Ras proteins to the plasma membrane and endomembranes through posttranslational addition of farnesyl and palmitoyl residues is an important mechanism through which cells provide specificity to Ras signal output. Although the Aspergillus fumigatus RasA protein is known to be a major regulator of growth and development, the membrane distribution of RasA during polarized morphogenesis and the role of properly localized Ras signaling in virulence of a pathogenic mold remain unknown. Here we demonstrate that Aspergillus fumigatus RasA localizes primarily to the plasma membrane of actively growing hyphae. We show that treatment with the palmitoylation inhibitor 2-bromopalmitate disrupts normal RasA plasma membrane association and decreases hyphal growth. Targeted mutations of the highly conserved RasA palmitoylation motif also mislocalized RasA from the plasma membrane and led to severe hyphal abnormalities, cell wall structural changes, and reduced virulence in murine invasive aspergillosis. Finally, we provide evidence that proper RasA localization is independent of the Ras palmitoyltransferase homolog, encoded by erfB, but requires the palmitoyltransferase complex subunit, encoded by erfD. Our results demonstrate that plasma membrane-associated RasA is critical for polarized morphogenesis, cell wall stability, and virulence in A. fumigatus.

  4. “Acting” on GLUT4: Membrane & Cytoskeletal Components of Insulin Action

    PubMed Central

    Brozinick, Joseph T.; Berkemeier, Bradley A.; Elmendorf, Jeffrey S.

    2008-01-01

    The dissection of mechanisms that regulate glucose transport by insulin has revealed an intricate network of signaling molecules scattered from the insulin receptor to the intracellular glucose transporter GLUT4. It is also appreciated that some insulin receptor signals jaunt in different directions to regulate events essential for the efficient redistribution of GLUT4 to the plasma membrane. Moreover key assists in the process appear to be arranged by membrane lipids and cytoskeletal proteins. Following current considerations of insulin signals regulating GLUT4, this review will focus on in vitro and in vivo evidence that supports an essential role for phosphoinositides and actin filaments in the control of glucose transport. The discussion will visit recent cell culture, whole animal, and human data highlighting membrane and cytoskeletal aspects of insulin resistance. PMID:18220662

  5. "Actin"g on GLUT4: membrane & cytoskeletal components of insulin action.

    PubMed

    Brozinick, Joseph T; Berkemeier, Bradley A; Elmendorf, Jeffrey S

    2007-05-01

    The dissection of mechanisms that regulate glucose transport by insulin has revealed an intricate network of signaling molecules scattered from the insulin receptor to the intracellular glucose transporter GLUT4. It is also appreciated that some insulin receptor signals jaunt in different directions to regulate events essential for the efficient redistribution of GLUT4 to the plasma membrane. Moreover key assists in the process appear to be arranged by membrane lipids and cytoskeletal proteins. Following current considerations of insulin signals regulating GLUT4, this review will focus on in vitro and in vivo evidence that supports an essential role for phosphoinositides and actin filaments in the control of glucose transport. The discussion will visit recent cell culture, whole animal, and human data highlighting membrane and cytoskeletal aspects of insulin resistance.

  6. Localization and Quantitation of Chloroplast Enzymes and Light-Harvesting Components Using Immunocytochemical Methods 12

    PubMed Central

    Mustardy, Laszlo; Cunningham, Francis X.; Gantt, Elisabeth

    1990-01-01

    Seven chloroplast proteins were localized in Porphyridium cruentum (ATCC 50161) by immunolabeling with colloidal gold on electron microscope sections of log phase cells grown under red, green, and white light. Ribulose bisphosphate carboxylase labeling occurred almost exclusively in the pyrenoid. The major apoproteins of photosystem I (56-64 kD) occurred mostly over the stromal thylakoid region and also appeared over the thylakoids passing through the pyrenoid. Labeling for photosystem II core components (D2 and a 45 kD Chl-binding protein), for phycobilisomes (allophycocyanin, and a 91 kD Lcm linker) and for ATP synthase (β subunit) were predominantly present in the thylakoid region but not in the pyrenoid region of the chloroplast. Red light cells had increased labeling per thylakoid length for polypeptides of photosystem II and of phycobilisomes, while photosystem I density decreased, compared to white light cells. Conversely, green light cells had a decreased density of photosystem II and phycobilisome polypeptides, while photosystem I density changed little compared with white light cells. A comparison of the immunogold labeling results with data from spectroscopic methods and from rocket immunoelectrophoresis indicates that it can provide a quantitative measure of the relative amounts of protein components as well as their localization in specific organellar compartments. Images Figure 1 Figure 2 PMID:16667706

  7. An optimized ensemble local mean decomposition method for fault detection of mechanical components

    NASA Astrophysics Data System (ADS)

    Zhang, Chao; Li, Zhixiong; Hu, Chao; Chen, Shuai; Wang, Jianguo; Zhang, Xiaogang

    2017-03-01

    Mechanical transmission systems have been widely adopted in most of industrial applications, and issues related to the maintenance of these systems have attracted considerable attention in the past few decades. The recently developed ensemble local mean decomposition (ELMD) method shows satisfactory performance in fault detection of mechanical components for preventing catastrophic failures and reducing maintenance costs. However, the performance of ELMD often heavily depends on proper selection of its model parameters. To this end, this paper proposes an optimized ensemble local mean decomposition (OELMD) method to determinate an optimum set of ELMD parameters for vibration signal analysis. In OELMD, an error index termed the relative root-mean-square error (Relative RMSE) is used to evaluate the decomposition performance of ELMD with a certain amplitude of the added white noise. Once a maximum Relative RMSE, corresponding to an optimal noise amplitude, is determined, OELMD then identifies optimal noise bandwidth and ensemble number based on the Relative RMSE and signal-to-noise ratio (SNR), respectively. Thus, all three critical parameters of ELMD (i.e. noise amplitude and bandwidth, and ensemble number) are optimized by OELMD. The effectiveness of OELMD was evaluated using experimental vibration signals measured from three different mechanical components (i.e. the rolling bearing, gear and diesel engine) under faulty operation conditions.

  8. Solubilization of pig lymphocyte plasma membrane and fractionation of some of the components

    PubMed Central

    Allan, D.; Crumpton, M. J.

    1971-01-01

    The degree of solubilization of pig lymphocyte plasma membrane by sodium deoxycholate was determined at a variety of temperatures and detergent concentrations. Approx. 95% of the membrane protein was soluble in 2% deoxycholate at 23°C. Some of the biological activities of the membrane survived this treatment. The leucine β-naphthylamidase activity was more readily soluble than the 5′-nucleotidase and these enzymes could be separated by extraction with 0.5% deoxycholate at 0°C. Membrane solubilized in 2% deoxycholate at 23°C was fractionated by sucrose-density-gradient centrifugation in 1% deoxycholate. The phospholipid was separated from the protein, which formed a fairly symmetrical peak that sedimented slightly slower than ovalbumin; the leucine naphthylamidase and 5′-nucleotidase activities were resolved from each other and from the main protein peak. Similar separations were achieved by elution from Sephadex G-200 and Sepharose 6B in 1% deoxycholate. The main proteins, however, appeared to possess much higher molecular weights than those indicated by sucrose-density-gradient centrifugation. This disparity suggests that many of the membrane proteins have a rod-like shape, especially since the results of experiments with [14C]deoxycholate revealed that the proteins did not bind significant amounts of deoxycholate. In contrast, 5′-nucleotidase and leucine naphthylamidase appeared to be globular. Polyacrylamide-gel electrophoresis of membrane solubilized in sodium dodecyl sulphate gave a similar distribution of protein to that achieved by sucrose-density-gradient centrifugation. Trace amounts only of polypeptides of molecular weight less than 10000 were detected. ImagesPLATE 1 PMID:4256533

  9. In vitro synthesis of immunoglobulins, secretory component and complement in normal and pathological skin and the adjacent mucous membranes

    PubMed Central

    Lai A Fat, R. F. M.; Suurmond, D.; Van Furth, R.

    1973-01-01

    A study on the synthesis of immunoglobulins (IgG, IgA, IgM, IgD, and IgE), secretory component and complement in normal and pathological skin and in the adjacent mucous membranes (i.e. conjunctiva, nasal, oral and vaginal mucosa) is reported. The results are based on the culture of tissue samples in a medium with two radioactive amino acids and the detection of synthesized proteins by autoradiography of the immunoelectrophoretic pattern of the culture fluid, except in the case of IgE for which the Ouchterlony technique was used. The results indicate that the normal skin does not synthesize immunoglobulins, whereas normal mucous membranes produce IgG and IgA. In the lesions of various skin diseases immunoglobulins are synthesized, mainly IgG but sometimes also IgA and IgE. The cells responsible for the production of immunoglobulins are plasma cells and lymphoid cells present in the skin lesions and mucous membranes. Synthesis of the free secretory component could be demonstrated only in certain mucous membranes (i.e. conjunctiva, nasal mucosa, and oral mucosa). Complement (C3) synthesis was found in normal skin, mucous membranes (i.e. conjunctiva, nasal and oral mucosa), and in the lesions of such skin diseases as discoid lupus erythematosus, (bullous) pemphigoid, dermatitis herpetiformis, malignant reticulosis, eczema and lichen planus. Complement production was also demonstrated in allergic skin reactions (i.e. tissue from allergic-positive patch tests, positive Mantoux tests and drug eruptions), but no immunoglobulin synthesis was detected in these lesions. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5 PMID:4199092

  10. Localization of Membrane-Associated Proteins in Vesicular Stomatitis Virus by Use of Hydrophobic Membrane Probes and Cross-Linking Reagents

    PubMed Central

    Zakowski, Jack J.; Wagner, Robert R.

    1980-01-01

    The location of membrane-associated proteins of vesicular stomatitis virus was investigated by using two monofunctional and three bifunctional probes that differ in the degree to which they partition into membranes and in their specific group reactivity. Two hydrophobic aryl azide probes, [125I]5-iodonaphthyl-1-azide and [3H]pyrenesulfonylazide, readily partitioned into virion membrane and, when activated to nitrenes by UV irradiation, formed stable covalent adducts to membrane constituents. Both of these monofunctional probes labeled the glyco-protein G and matrix M proteins, but [125I]5-iodonaphthyl-1-azide also labeled the nucleocapsid N protein and an unidentified low-molecular-weight component. Protein labeling of intact virions was unaffected by the presence of cytochrome c or glutathione, but disruption of membrane by sodium dodecyl sulfate greatly enhanced the labeling of all viral proteins except G. Labeling of G protein was essentially restricted to the membrane-embedded, thermolysin-resistant tail fragment. Three bifunctional reagents, tartryl diazide, dimethylsuberimidate, and 4,4′-dithiobisphenylazide, were tested for their capacity to cross-link proteins to membrane phospholipids of virions grown in the presence of [3H]palmitate. Only G and M proteins of intact virions were labeled with 3H-phospholipid by these cross-linkers; the reactions were not affected by cytochrome c but were abolished by disruption of virus with sodium dodecyl sulfate. Dimethylsuberimidate, which reacts with free amino groups, cross-linked 3H-phospholipid to both G and M protein. In contrast, the hydrophilic tartryl diazide cross-linked phospholipid primarily to the M protein, whereas the hydrophobic 4,4′-dithiobisphenylazide cross-linked phospholipid primarily to the intrinsic G protein. These data support the hypothesis that the G protein traverses the virion membrane and that the M protein is membrane associated but does not penetrate very deeply, if at all. PMID:6255216

  11. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    NASA Astrophysics Data System (ADS)

    Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

    2010-03-01

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  12. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

    PubMed

    Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

    2010-03-19

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  13. Ultrastructural localization of cytochrome b in the membranes of resting and phagocytosing human granulocytes.

    PubMed Central

    Jesaitis, A J; Buescher, E S; Harrison, D; Quinn, M T; Parkos, C A; Livesey, S; Linner, J

    1990-01-01

    Affinity-purified rabbit anti-neutrophil cytochrome b light or heavy chain antibodies were used to immunocytochemically and biochemically localize cytochrome b in neutrophils and eosinophils. The antibodies were monospecific, recognizing polypeptides of 91 and 22 kD, respectively, on Western blots of whole neutrophil extracts. The antibodies were used in Western blot analysis of subcellular fractions of purified neutrophils to confirm that the distribution of cytochrome b spectral absorbance matched that of the two subunits. Thin sections of cryofixed, molecular distillation-dried granulocytes were labeled with the anti-cytochrome b antibodies, followed by incubation with biotin-conjugated secondary antibody, and final labeling with streptavidin-conjugated colloidal gold. Electron microscopy revealed that the cytochrome b light and heavy chains were localized primarily (80%) to 0.1-0.2-micron round or elliptical granule-like structures in neutrophils and 0.4-0.5-micron granules in eosinophils. Approximately 20% of the cytochrome b was localized to the surface, confirming the subcellular fractionation studies. Double staining experiments on the neutrophils, using polyclonal rabbit anti-lactoferrin antibody, indicated that the cytochrome-bearing structures also contained lactoferrin and thus were specific granules. When the analysis was performed on neutrophils that had phagocytosed Staphylococcus aureus, cytochrome b was found in the phagosomal membrane adjoining the bacterial cell wall. Images PMID:2312727

  14. Transport mechanism for succinate and phosphate localized in the plasma membrane of bovine spermatozoa.

    PubMed

    Babcock, D F; First, N L; Lardy, H A

    1975-08-25

    Bovine spermatozoa accumulated a small amount of 32Pi during aerobic incubation in vitro. At least 50% of the acquired isotope rapidly entered cellular nucleotides. Both adenosine and guanosine di- and triphosphates were labeled, but contrary to expectations, the specific activity of ADP exceeded that of ATP. The uptake of phosphate and its incorporation into nucleotides were suppressed by respiratory inhibitors and were abolished by treatment with sulfhydryl-directed reagents at 10 to 20 nmol/mg of sperm protein. With fructose as an energy source for motility, glycolysis did not support phosphate uptake. Nucleotide labeling was increased 60 to 80-fold when the cells were treated with the polyene antibiotic filipin, and filipin was able to reverse the inhibition of phosphate (and succinate) entry produced by N-ethylmaleimide or mersalyl. Since filipin interacts specifically with the cholesterol-containing plasma membrane of bovine spermatozoa and increases its permeability, it is probable that the plasma membrane normally limits phosphate and succinate transport into these cells. This contention is further supported by the observation that high concentrations of extracellular Pi, the penetration of which was extremely limited under these conditions, protected against inactivation by N-ethylmaleimide. Phosphate uptake was increased 10 to 20-fold, but nucleotide labeling was inhibited, when calcium was present in the incubation medium. Ruthenium red, presumably acting extracellularly, prevented these effects of calcium. Thus, the entry of phosphate and succinate into spermatozoa is controlled by plasma membrane components that resemble the phosphate and succinate exchangers and calcium carrier found in mitochondria isolated from other sources.

  15. Externally applied cyclic strain regulates localization of focal contact components in cultured smooth muscle cells.

    PubMed

    Cunningham, James J; Linderman, Jennifer J; Mooney, David J

    2002-01-01

    Mechanical signals are critical regulators of cellular gene expression, yet little is understood of the mechanism whereby cells sense mechanical forces. In this study we have tested the hypothesis that mechanical strain applied to populations of cells via their adhesion substrate rapidly alters the cellular distribution of focal contact proteins. Focal contact-associated components (vinculin, a-actinin, paxillin) were assayed by immunofluorescence microscopy and quantitative western blotting. Application of a single step increase in strain in multiple experiments caused overall a small change in focal contact-associated vinculin. In contrast, cyclic strain induced a large and very reproducible increase in detergent-insoluble vinculin (52% relative to static) after just 1 min of strain. Insoluble paxillin was transiently enriched with a similar time course, whereas insoluble a-actinin did not change significantly in response to cyclic strain. Rhodamine-labeled chicken vinculin added to permeabilized cells preferentially localized to focal contacts in response to cyclic strain, but not a single step increase in strain. These findings establish that insoluble levels of focal contact components are altered rapidly following application of an appropriate number of mechanical perturbations, and suggest that at least one component of the mechanism does not involve soluble intermediates.

  16. Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in "Bacillus Subtilis"

    ERIC Educational Resources Information Center

    Bredeston, L. M.; Marciano, D.; Albanesi, D.; De Mendoza, D.; Delfino, J. M.

    2011-01-01

    This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of "Bacillus subtilis", composed of a [delta]5-desaturase (encoded by the "des" gene) and the canonical…

  17. Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in "Bacillus Subtilis"

    ERIC Educational Resources Information Center

    Bredeston, L. M.; Marciano, D.; Albanesi, D.; De Mendoza, D.; Delfino, J. M.

    2011-01-01

    This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of "Bacillus subtilis", composed of a [delta]5-desaturase (encoded by the "des" gene) and the canonical…

  18. Detecting and localizing failure points in proton exchange membrane fuel cells using IR thermography

    NASA Astrophysics Data System (ADS)

    Bender, Guido; Felt, Wyatt; Ulsh, Michael

    2014-05-01

    An understanding of the potentially serious long-term performance degradation effects that coating and/or other fabrication irregularities might have in mass produced proton exchange membrane fuel cells (PEMFC) is essential to determine manufacturing tolerances of fuel cell components. An experimental setup and methodology is described that employs accelerated stress tests (ASTs) and IR thermography to accurately determine the location and severity of developing failure points in PEMFCs. The method entails a novel hardware that allows the spatial observation of a hydrogen crossover experiment within a fuel cell hardware. The application of the method is demonstrated by comparing the effects of an AST on pristine as well as defect-containing MEAs. The presented method is shown to be valuable for determining the areas within a fuel cell that are most stressed by aging processes.

  19. Progesterone receptor membrane component 1 (PGRMC1) expression in fetal membranes among women with preterm premature rupture of the membranes (PPROM).

    PubMed

    Feng, L; Antczak, B C; Lan, L; Grotegut, C A; Thompson, J L; Allen, T K; Murtha, A P

    2014-05-01

    PGRMC1 function is implicated in maintaining fetal membrane (FM) integrity. PGRMC1 was detectable primarily in the cytoplasm of FM cells and was actively regulated in FMs and relevant for PGRMC1-mediated progesterone action. By cell type, PGRMC1 expression was higher in amnion and chorion compared with decidua. By clinical phenotype, PGRMC1 expression was higher among preterm-no-labor and term-no-labor subjects compared to PPROM. PGRMC1 expression appears to be diminished in PPROM subjects. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    SciTech Connect

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

    1990-02-13

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

  1. Selective excitation for spectral editing and assignment in separated local field experiments of oriented membrane proteins

    NASA Astrophysics Data System (ADS)

    Koroloff, Sophie N.; Nevzorov, Alexander A.

    2017-01-01

    Spectroscopic assignment of NMR spectra for oriented uniformly labeled membrane proteins embedded in their native-like bilayer environment is essential for their structure determination. However, sequence-specific assignment in oriented-sample (OS) NMR is often complicated by insufficient resolution and spectral crowding. Therefore, the assignment process is usually done by a laborious and expensive "shotgun" method involving multiple selective labeling of amino acid residues. Presented here is a strategy to overcome poor spectral resolution in crowded regions of 2D spectra by selecting resolved "seed" residues via soft Gaussian pulses inserted into spin-exchange separated local-field experiments. The Gaussian pulse places the selected polarization along the z-axis while dephasing the other signals before the evolution of the 1H-15N dipolar couplings. The transfer of magnetization is accomplished via mismatched Hartmann-Hahn conditions to the nearest-neighbor peaks via the proton bath. By optimizing the length and amplitude of the Gaussian pulse, one can also achieve a phase inversion of the closest peaks, thus providing an additional phase contrast. From the superposition of the selective spin-exchanged SAMPI4 onto the fully excited SAMPI4 spectrum, the 15N sites that are directly adjacent to the selectively excited residues can be easily identified, thereby providing a straightforward method for initiating the assignment process in oriented membrane proteins.

  2. Selective excitation for spectral editing and assignment in separated local field experiments of oriented membrane proteins.

    PubMed

    Koroloff, Sophie N; Nevzorov, Alexander A

    2017-01-01

    Spectroscopic assignment of NMR spectra for oriented uniformly labeled membrane proteins embedded in their native-like bilayer environment is essential for their structure determination. However, sequence-specific assignment in oriented-sample (OS) NMR is often complicated by insufficient resolution and spectral crowding. Therefore, the assignment process is usually done by a laborious and expensive "shotgun" method involving multiple selective labeling of amino acid residues. Presented here is a strategy to overcome poor spectral resolution in crowded regions of 2D spectra by selecting resolved "seed" residues via soft Gaussian pulses inserted into spin-exchange separated local-field experiments. The Gaussian pulse places the selected polarization along the z-axis while dephasing the other signals before the evolution of the (1)H-(15)N dipolar couplings. The transfer of magnetization is accomplished via mismatched Hartmann-Hahn conditions to the nearest-neighbor peaks via the proton bath. By optimizing the length and amplitude of the Gaussian pulse, one can also achieve a phase inversion of the closest peaks, thus providing an additional phase contrast. From the superposition of the selective spin-exchanged SAMPI4 onto the fully excited SAMPI4 spectrum, the (15)N sites that are directly adjacent to the selectively excited residues can be easily identified, thereby providing a straightforward method for initiating the assignment process in oriented membrane proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites

    PubMed Central

    Park, Jae-Sook; Thorsness, Mary K.; Policastro, Robert; McGoldrick, Luke L.; Hollingsworth, Nancy M.; Thorsness, Peter E.; Neiman, Aaron M.

    2016-01-01

    The Vps13 protein family is highly conserved in eukaryotic cells. Mutations in human VPS13 genes result in a variety of diseases, such as chorea acanthocytosis (ChAc), but the cellular functions of Vps13 proteins are not well defined. In yeast, there is a single VPS13 orthologue, which is required for at least two different processes: protein sorting to the vacuole and sporulation. This study demonstrates that VPS13 is also important for mitochondrial integrity. In addition to preventing transfer of DNA from the mitochondrion to the nucleus, VPS13 suppresses mitophagy and functions in parallel with the endoplasmic reticulum–mitochondrion encounter structure (ERMES). In different growth conditions, Vps13 localizes to endosome–mitochondrion contacts and to the nuclear–vacuole junctions, indicating that Vps13 may function at membrane contact sites. The ability of VPS13 to compensate for the absence of ERMES correlates with its intracellular distribution. We propose that Vps13 is present at multiple membrane contact sites and that separation-of-function mutants are due to loss of Vps13 at specific junctions. Introduction of VPS13A mutations identified in ChAc patients at cognate sites in yeast VPS13 are specifically defective in compensating for the lack of ERMES, suggesting that mitochondrial dysfunction might be the basis for ChAc. PMID:27280386

  4. Endogenous Galectin-3 Is Localized in Membrane Lipid Rafts and Regulates Migration of Dendritic Cells

    PubMed Central

    Hsu, Daniel K.; Chernyavsky, Alexander I.; Chen, Huan-Yuan; Yu, Lan; Grando, Sergei A.; Liu, Fu-Tong

    2008-01-01

    This study reveals a function of endogenous galectin-3, an animal lectin recognizing β-galactosides, in regulating dendritic cell motility both in vitroand in vivo,which to our knowledge is unreported. First, galectin-3-deficient (gal3−/−) bone marrow-derived dendritic cells exhibited defective chemotaxis compared to gal3+/+ cells. Second, cutaneous dendritic cells in gal3−/− mice displayed reduced migration to draining lymph nodes upon hapten stimulation compared to gal3+/+ mice. Moreover, gal3−/− mice were impaired in the development of contact hypersensitivity relative to gal3+/+ mice in response to a hapten, a process in which dendritic cell trafficking to lymph nodes is critical. In addition, defective signaling was detected in gal3−/− cells upon chemokine receptor activation. By immunofluorescence microscopy, we observed that galectin-3 is localized in membrane ruffles and lamellipodia in stimulated dendritic cells and macrophages. Furthermore, galectin-3 was enriched in lipid raft domains under these conditions. Finally, we determined that ruffles on gal3−/− cells contained structures with lower complexity compared to gal3+/+ cells. In view of the participation of membrane ruffles in signal transduction and cell motility, we conclude that galectin-3 regulates cell migration by functioning at these structures. PMID:18843294

  5. Gastrointestinal Hormone Cholecystokinin Increases P-Glycoprotein Membrane Localization and Transport Activity in Caco-2 Cells.

    PubMed

    Yano, Kentaro; Shimizu, Saori; Tomono, Takumi; Ogihara, Takuo

    2017-09-01

    It was reported that stimulation of taste receptor type 2 member 38 by a bitter substance, phenylthiocarbamide (PTC), increased P-glycoprotein (P-gp) mRNA level and transport activity via release of the gastrointestinal hormone cholecystokinin-8 (CCK-8) at 9 h. Therefore, we hypothesized that CCK-8 and PTC might also regulate P-gp activity more rapidly via a different mechanism. As a result, we found that the pretreatment of human colon adenocarcinoma (Caco-2) cells with 10-mM PTC significantly decreased the intracellular accumulation of P-gp substrate rhodamine 123 (Rho123) compared with the control after 90-min incubation. Moreover, CCK-8 treatments significantly reduced the accumulation of Rho123 within 30 min, compared with the control. On the other hand, when Caco-2 cells were pretreated with PTC, the efflux ratio of Rho123 was significantly increased compared with control. The efflux ratio of Rho123 in CCK-8 treatment cells was also significantly increased compared with control. Furthermore, CCK-8 increased the phosphorylation of the scaffold proteins ezrin, radixin, and moesin, which regulate translocation of P-gp to the plasma membrane. Therefore, our results indicate that PTC induced release of CCK-8, which in turn induced the phosphorylation of ezrin, radixin, and moesin proteins, leading to upregulation of P-gp transport activity via increased membrane localization of P-gp. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  6. In-situ Monitoring of Internal Local Temperature and Voltage of Proton Exchange Membrane Fuel Cells

    PubMed Central

    Lee, Chi-Yuan; Fan, Wei-Yuan; Hsieh, Wei-Jung

    2010-01-01

    The distribution of temperature and voltage of a fuel cell are key factors that influence performance. Conventional sensors are normally large, and are also useful only for making external measurements of fuel cells. Centimeter-scale sensors for making invasive measurements are frequently unable to accurately measure the interior changes of a fuel cell. This work focuses mainly on fabricating flexible multi-functional microsensors (for temperature and voltage) to measure variations in the local temperature and voltage of proton exchange membrane fuel cells (PEMFC) that are based on micro-electro-mechanical systems (MEMS). The power density at 0.5 V without a sensor is 450 mW/cm2, and that with a sensor is 426 mW/cm2. Since the reaction area of a fuel cell with a sensor is approximately 12% smaller than that without a sensor, but the performance of the former is only 5% worse. PMID:22163556

  7. In-situ monitoring of internal local temperature and voltage of proton exchange membrane fuel cells.

    PubMed

    Lee, Chi-Yuan; Fan, Wei-Yuan; Hsieh, Wei-Jung

    2010-01-01

    The distribution of temperature and voltage of a fuel cell are key factors that influence performance. Conventional sensors are normally large, and are also useful only for making external measurements of fuel cells. Centimeter-scale sensors for making invasive measurements are frequently unable to accurately measure the interior changes of a fuel cell. This work focuses mainly on fabricating flexible multi-functional microsensors (for temperature and voltage) to measure variations in the local temperature and voltage of proton exchange membrane fuel cells (PEMFC) that are based on micro-electro-mechanical systems (MEMS). The power density at 0.5 V without a sensor is 450 mW/cm(2), and that with a sensor is 426 mW/cm(2). Since the reaction area of a fuel cell with a sensor is approximately 12% smaller than that without a sensor, but the performance of the former is only 5% worse.

  8. Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells.

    PubMed

    Dyrda, Agnieszka; Cytlak, Urszula; Ciuraszkiewicz, Anna; Lipinska, Agnieszka; Cueff, Anne; Bouyer, Guillaume; Egée, Stéphane; Bennekou, Poul; Lew, Virgilio L; Thomas, Serge L Y

    2010-02-26

    The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+) and Cl(-) currents were strictly dependent on the presence of Ca(2+). The Ca(2+)-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+) permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca(2+) permeability pathway leading to increased [Ca(2+)](i), secondary activation of Ca(2+)-sensitive K(+) channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias

  9. Membrane Localization of the S1 Subunit of Pertussis Toxin in Bordetella pertussis and Implications for Pertussis Toxin Secretion

    PubMed Central

    Farizo, Karen M.; Fiddner, Stefanie; Cheung, Anissa M.; Burns, Drusilla L.

    2002-01-01

    Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system. PMID:11854200

  10. Characterization of pneumolysin from Streptococcus pneumoniae, interacting with carbohydrate moiety and cholesterol as a component of cell membrane.

    PubMed

    Lim, Jong Eun; Park, Seong Ah; Bong, Seoung Min; Chi, Young Min; Lee, Ki Seog

    2013-01-11

    The cytolytic mechanism of cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target cell membrane. Membrane cholesterol was thought to serve as the common receptor for these toxins, but not all CDCs require cholesterol for binding. One member of this toxin family, pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae, and the mechanism via which PLY binds to its putative receptor or cholesterol on the cell membrane is still poorly understood. Here, we demonstrated that PLY interacted with carbohydrate moiety and cholesterol as a component of the cell membrane, using the inhibitory effect of hemolytic activity. The hemolytic activity of PLY was inhibited by cholesterol-MβCD, which is in a 3β configuration at the C3-hydroxy group, but is not in a 3α-configuration. In the interaction between PLY and carbohydrate moiety, the mannose showed a dose-dependent increase in the inhibition of PLY hemolytic activity. The binding ability of mannose with truncated PLYs, as determined by the pull-down assay, showed that mannose might favor binding to domain 4 rather than domains 1-3. These studies provide a new model for the mechanism of cellular recognition by PLY, as well as a foundation for future investigations into whether non-sterol molecules can serve as receptors for other members of the CDC family of toxins. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

    PubMed

    Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

    2017-04-28

    The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Activation of cell membrane-localized Toll-like receptor 3 by siRNA.

    PubMed

    Pirher, Nina; Pohar, Jelka; Manček-Keber, Mateja; Benčina, Mojca; Jerala, Roman

    2017-09-01

    Small interfering RNA molecules (siRNA) are short dsRNAs that are used for different therapeutic applications. On the other hand, dsRNAs can bind to and activate cell RNA sensors and consequently trigger inflammatory response. Here we show that siRNA activates primary human endothelial cells and human lymphatic endothelial cells and that this response is inhibited by antibodies against TLR3. In contrast, the activation of human lymphatic endothelial cells by poly(I:C) was inhibited by bafilomycin but not by anti-TLR3 antibodies. Bafilomycin also inhibited poly(I:C) but not siRNA cell stimulation in TLR3-transfected HEK293. The response to siRNA required the expression of UNC93B1, which directs TLR3 to the surface of HEK293 cells. We propose that the engaged signaling pathway of TLR3 depends on the receptor localization and on the length of the dsRNA, where the activation of cell membrane TLR3 by short dsRNA leads to a predominantly proinflammatory response, whereas TLR3 activation in endosomal compartments by long dsRNA is characterized by the production of type I IFN. A molecular model suggests that the siRNA can bind to the binding sites of the TLR3 ectodomain and trigger receptor dimerization. These results contribute to understanding of the mechanism of side effects seen in the therapeutic application of naked, unmodified siRNA as a result of the activation of TLR3 localized at the plasma membrane. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  13. Assessment of the local windblown component of dust in the western United States

    NASA Astrophysics Data System (ADS)

    Kavouras, Ilias G.; Etyemezian, Vicken; Xu, Jin; Dubois, David W.; Green, Mark; Pitchford, Marc

    2007-04-01

    We estimated the contributions of windblown dust from nearby area sources to dust concentrations at Class I areas in the western United States including Alaska and Hawaii. The approach utilized multivariate linear regression of dust concentrations against categorized wind conditions (wind direction and speed) for all 2001-2003 data for 70 Interagency Monitoring of Protected Visual Environments sites. Statistically significant associations between dust concentrations and at least one of the wind variables were found at 41 sites with correlation coefficients as high as 0.97. At some sites, primarily in New Mexico and Texas, windblown dust from nearby sources accounted for up to 3 μg m-3 over the 2001-2003 period. In addition, the impact of local windblown dust sources during the 20% worst visibility days when dust was the major component of visibility reduction (worst dust days) was examined. A total of 608 worst dust days were identified for 2001-2003, mostly at Class I areas in southwestern states during spring and summer with 24-h average dust concentrations as high as 153 μg m-3. Windblown dust from local sources was present with statistical confidence on many of the worst dust days at sites in New Mexico, Utah, Colorado, southern Texas, and Death Valley in California. A smaller percentage of worst dust days were associated with local windblown dust in Arizona and other sites in southern California, suggesting either nonwindblown or distant sources of dust. The methods discussed can serve as a useful, semiquantitative tool for identifying sites where local wind conditions affect dust concentrations.

  14. Localization of the mei-1 gene product of Caenorhaditis elegans, a meiotic-specific spindle component

    PubMed Central

    1994-01-01

    Genetic evidence suggests that the product of the mei-1 gene of Caenorhabditis elegans is specifically required for meiosis in the female germline. Loss-of-function mei-1 mutations block meiotic spindle formation while a gain-of-function allele instead results in spindle defects during the early mitotic cleavages. In this report, we use immunocytochemistry to examine the localization of the mei-1 product in wild-type and mutant embryos. During metaphase of meiosis I in wild- type embryos, mei-1 protein was found throughout the spindle but was more concentrated toward the poles. At telophase I, mei-1 product colocalized with the chromatin at the spindle poles. The pattern was repeated during meiosis II but no mei-1 product was visible during the subsequent mitotic cleavages. The mei-1 gain-of-function allele resulted in ectopic mei-1 staining in the centers of the microtubule- organizing centers during interphase and in the spindles during the early cleavages. This aberrant localization is probably responsible for the poorly formed and misoriented cleavage spindles characteristic of the mutation. We also examined the localization of mei-1(+) product in the presence of mutations of genes that genetically interact with mei-1 alleles. mei-2 is apparently required to localize mei-1 product to the spindle during meiosis while mel-26 acts as a postmeiotic inhibitor. We conclude that mei-1 encodes a novel spindle component, one that is specialized for the acentriolar meiotic spindles unique to female meiosis. The genes mei-2 and mel-26 are part of a regulatory network that confines mei-1 activity to meiosis. PMID:8027178

  15. [Optimization of labeling and localizing bacterial membrane and nucleus with FM4-64 and Hoechst dyes].

    PubMed

    Wang, Jing; Han, Yanping; Yang, Ruifu; Zhao, Xingxu

    2015-08-04

    To observe cell membrane and nucleus in bacteria for subcellular localization. FM4-64 and Hoechst were dyed that can label cell membrane and nucleus, respectively. Both dyes were used to co-stain the membranes and nucleus of eight bacterial strains ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Yersinia pestis, Legionella pneumonia, Vibrio cholerae and Bacillus anthracis). E. coli was dyed with different dye concentrations and times and then observed by confocal fluorescence microscopic imaging. Fluorescence intensity of cell membrane and nucleus is affected by dye concentrations and times. The optimal conditions were determined as follows: staining cell membrane with 20 μg/mL FM4-64 for 1 min and cell nucleus with 20 μg/mL Hoechst for 20 min. Gram-negative bacteria were dyed better than gram-positive bacteria with FM4-64dye. FM4-64 and Hoechst can be used to stain membrane and nucleus in different types of bacteria. Co-staining bacterial membrane and nucleus provides the reference to observe cell structure in prokaryotes for studying subcellular localization.

  16. [Localization of ATPase activity, respiration and ultrastructure of wheat root cells with modulated ion conductivity of plasma membrane].

    PubMed

    Chernysheva, F A; Alekseeva, V Ia; Polygalova, O O; Gordon, L Kh

    2004-01-01

    Changes in the localization of ATPase activity, respiration and ultrastructure of wheat root cells with modulated ion conductivity of plasma membrane were studied. A 2 h treatment of excised root with valinomycin (20 microM), N,N-dicyclohexylcarbodiimid (100 microM), gramicidin S (20 microM) and chlorpromazine (100 microM) caused an increased loss of potassium by cells, a decreased respiration and changes in the localization of ATPase activity and in cell ultrastructure. Differences in the observed changes may be conditioned by different mechanisms of action of the membrane active compounds used. It is concluded that changes in the localization of ATPase activity and ultrastructure may indicate some early specific responses of root cells, whereas the increase in the ion conductivity and decrease in respiration under disruption of ion homeostasis caused by membrane active compounds indicate unspecific responses of cells.

  17. Phase-coherence classification: A new wavelet-based method to separate local field potentials into local (in)coherent and volume-conducted components.

    PubMed

    von Papen, M; Dafsari, H; Florin, E; Gerick, F; Timmermann, L; Saur, J

    2017-11-01

    Local field potentials (LFP) reflect the integrated electrophysiological activity of large neuron populations and may thus reflect the dynamics of spatially and functionally different networks. We introduce the wavelet-based phase-coherence classification (PCC), which separates LFP into volume-conducted, local incoherent and local coherent components. It allows to compute power spectral densities for each component associated with local or remote electrophysiological activity. We use synthetic time series to estimate optimal parameters for the application to LFP from within the subthalamic nucleus of eight Parkinson patients. With PCC we identify multiple local tremor clusters and quantify the relative power of local and volume-conducted components. We analyze the electrophysiological response to an apomorphine injection during rest and hold. Here we show medication-induced significant decrease of incoherent activity in the low beta band and increase of coherent activity in the high beta band. On medication significant movement-induced changes occur in the high beta band of the local coherent signal. It increases during isometric hold tasks and decreases during phasic wrist movement. The power spectra of local PCC components is compared to bipolar recordings. In contrast to bipolar recordings PCC can distinguish local incoherent and coherent signals. We further compare our results with classification based on the imaginary part of coherency and the weighted phase lag index. The low and high beta band are more susceptible to medication- and movement-related changes reflected by incoherent and local coherent activity, respectively. PCC components may thus reflect functionally different networks. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Molecular Architecture of the Major Membrane Ring Component of the Nuclear Pore Complex.

    PubMed

    Upla, Paula; Kim, Seung Joong; Sampathkumar, Parthasarathy; Dutta, Kaushik; Cahill, Sean M; Chemmama, Ilan E; Williams, Rosemary; Bonanno, Jeffrey B; Rice, William J; Stokes, David L; Cowburn, David; Almo, Steven C; Sali, Andrej; Rout, Michael P; Fernandez-Martinez, Javier

    2017-03-07

    The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the ∼120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.

  19. Immunocytochemical localization of sodium channel distributions in the excitable membranes of Electrophorus electricus.

    PubMed Central

    Ellisman, M H; Levinson, S R

    1982-01-01

    The tetrodotoxin binding protein, a major component of the Na+ channel, has been purified from the electric organ of the South American eel Electrophorus electricus. Antibodies to this protein were raised in rabbits and their specificity was demonstrated by a highly sensitive radioimmunoassay and by immunoprecipitation procedures. These antibodies were used to examine the distribution of the binding protein in the eel electroplax membranes and along myelinated nerve axons. The distribution of the antigen was determined by using the peroxidase-antiperoxidase technique at both the light and electron microscopic levels. In the electrocytes of the electric organ, only the innervated face showed staining in experimental material. The stained regions of electroplax plasmalemma included the caveolae of the innervated surface while caveolae of the non-innervated surface did not stain. Thus, the innervated surface including caveolae exclusively contains the Na+ channels. Along myelinated axons, staining was limited to the nodal zone of the node of Ranvier. The paranodal and internodal zones did not stain for the binding protein. Limited diffusion of primary IgG and subsequent reactants into the paranodal and internodal sites was eliminated as a possible source of focal staining at nodes because mechanically demyelinated preparations also exhibited focal nodal staining. Thus, this tetrodotoxin binding protein component of the Na+ channel is located solely within the nodal zone of the node of Ranvier. Images PMID:6292913

  20. One of the origins of plasma membrane phosphatidylserine in plant cells is a local synthesis by a serine exchange activity.

    PubMed

    Vincent, P; Maneta-Peyret, L; Sturbois-Balcerzak, B; Duvert, M; Cassagne, C; Moreau, P

    1999-12-24

    In plant cells, as in animal cells, the endoplasmic reticulum (ER) is considered to be the major site of phospholipid synthesis, and it has been shown that phosphatidylserine (PS) reaches the plasma membrane via the vesicular ER-Golgi-plasma membrane pathway in leek cells. However, it has never been determined whether the plasma membrane of leek cells is able to synthesize PS. We have analyzed the distribution of PS synthesizing enzymes along the vesicular pathway. In ER, Golgi and plasma membrane fractions isolated from leek cells, we have measured the activity of the two biosynthetic pathways leading to the synthesis of PS, i.e. serine exchange and CTP cytidylyltransferase plus PS synthase. We have found a high serine exchange activity in the plasma membrane fraction, and then determined that this membrane is able to synthesize both long chain fatty acid- and very long chain fatty acid-containing PS. Therefore, the PS in the plasma membrane of leek cells has two different origins: the intracellular vesicular pathway from the ER and a local synthesis in the plasma membrane.

  1. The Effect of Extracellular Components from Colletotrichum lindemuthianum on Membrane Transport in Vesicles Isolated from Bean Hypocotyl 1

    PubMed Central

    Rogers, Kim R.; Anderson, Anne J.

    1987-01-01

    Extracellular components released from mycelia of the α and β races of the bean pathogen, Colletotrichum lindemuthianum, inhibited proton uptake in sealed vesicles prepared from bean hypocotyls. Differential sensitivity of ATP-driven proton transport to nitrate, vanadate, N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and oligomycin suggested the vesicles were enriched for tonoplast. Anion stimulation of proton transport, by enhancement of ATPase activity and dissipation of the membrane potential, was consistent with this conclusion. Although fungal components inhibited the formation of a pH gradient, the membrane potential was unaffected and the ATPase activity slightly stimulated. These data suggest that the fungal components produce an electroneutral proton exchange. Proton transport in Dark Red Kidney bean tonoplast vesicles was inhibited by mycelial preparations from the incompatible α race and compatible β race. Elicitor activity, however, was greater in the α race fractions. Elicitor purified from α race culture filtrate did not inhibit proton transport in vesicles isolated from Dark Red Kidney bean. Consequently, elicitor activity need not be associated with an ability to impair tonoplast function. PMID:16665456

  2. The Effect of Extracellular Components from Colletotrichum lindemuthianum on Membrane Transport in Vesicles Isolated from Bean Hypocotyl.

    PubMed

    Rogers, K R; Anderson, A J

    1987-06-01

    Extracellular components released from mycelia of the alpha and beta races of the bean pathogen, Colletotrichum lindemuthianum, inhibited proton uptake in sealed vesicles prepared from bean hypocotyls. Differential sensitivity of ATP-driven proton transport to nitrate, vanadate, N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, and oligomycin suggested the vesicles were enriched for tonoplast. Anion stimulation of proton transport, by enhancement of ATPase activity and dissipation of the membrane potential, was consistent with this conclusion. Although fungal components inhibited the formation of a pH gradient, the membrane potential was unaffected and the ATPase activity slightly stimulated. These data suggest that the fungal components produce an electroneutral proton exchange. Proton transport in Dark Red Kidney bean tonoplast vesicles was inhibited by mycelial preparations from the incompatible alpha race and compatible beta race. Elicitor activity, however, was greater in the alpha race fractions. Elicitor purified from alpha race culture filtrate did not inhibit proton transport in vesicles isolated from Dark Red Kidney bean. Consequently, elicitor activity need not be associated with an ability to impair tonoplast function.

  3. Neural Processes in the Human Temporoparietal Cortex Separated by Localized Independent Component Analysis.

    PubMed

    Igelström, Kajsa M; Webb, Taylor W; Graziano, Michael S A

    2015-06-24

    The human temporoparietal junction (TPJ) is a topic of intense research. Imaging studies have identified TPJ activation in association with many higher-order functions such as theory-of-mind, episodic memory, and attention, causing debate about the distribution of different processes. One major challenge is the lack of consensus about the anatomical location and extent of the TPJ. Here, we address this problem using data-driven analysis to test the hypothesis that the bilateral TPJ can be parcellated into subregions. We applied independent component analysis (ICA) to task-free fMRI data within a local region around the bilateral TPJ, iterating the ICA at multiple model orders and in several datasets. The localized analysis allowed finer separation of processes and the use of multiple dimensionalities provided qualitative information about lateralization. We identified four subdivisions that were bilaterally symmetrical and one that was right biased. To test whether the independent components (ICs) reflected true subdivisions, we performed functional connectivity analysis using the IC coordinates as seeds. This confirmed that the subdivisions belonged to distinct networks. The right-biased IC was connected with a network often associated with attentional processing. One bilateral subdivision was connected to sensorimotor regions and another was connected to auditory regions. One subdivision that presented as distinct left- and right-biased ICs was connected to frontoparietal regions. Another subdivision that also had left- and right-biased ICs was connected to social or default mode networks. Our results show that the TPJ in both hemispheres hosts multiple neural processes with connectivity patterns consistent with well developed specialization and lateralization.

  4. Regulation of CED-3 caspase localization and activation by C. elegans nuclear membrane protein NPP-14

    PubMed Central

    Chen, Xudong; Wang, Yue; Chen, Yu-Zen; Harry, Brian L.; Nakagawa, Akihisa; Lee, Eui-Seung; Guo, Hongyan; Xue, Ding

    2017-01-01

    Caspases are cysteine proteases with critical roles in apoptosis. The Caenorhabditis elegans caspase CED-3 is activated by autocatalytic cleavage, a process enhanced by CED-4. Here we report that CED-3 zymogen localizes to the perinuclear region in C. elegans germ cells and that CED-3 autocatalytic cleavage is held in check by C. elegans nuclei and activated by CED-4. The nuclear pore protein NPP-14 interacts with the prodomain of CED-3 zymogen, colocalizes with CED-3 in vivo, and inhibits CED-3 autoactivation in vitro. Several missense mutations in the CED-3 prodomain result in stronger association with NPP-14 and reduced CED-3 activation by CED-4 in the presence of nuclei or NPP-14, leading to cell death defects. Those same mutations enhance autocatalytic cleavage of CED-3 in vitro and increase apoptosis in vivo in the absence of npp-14. Our results reveal a critical role for nuclei and nuclear membrane proteins in regulating activation and localization of CED-3. PMID:27723735

  5. How Local Anesthetics affect the structural and dynamical properties of bio-membranes

    NASA Astrophysics Data System (ADS)

    Yi, Zheng; Nagao, Michihiro; Bossev, Dobrin

    2009-03-01

    To address the question of how local anesthetics influences the structural and dynamical properties of bio-membranes, neutron-spin echo spectroscopy (NSE) has been performed on 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) unilamellar vesicles (ULV) with different concentrations of Lidocaine in D2O to study the influence of Lidocaine on the bending elasticity of DMPC ULV bilayers in fluid crystal (L&_slash;alpha) phase and the ripple gel (P&_slash;beta') phase; The measurement of small-angle neutron scattering (SANS) has been performed to determine the bilayer thickness as a function of the concentration of Lidocaine. In the existence of molecules of Lidocaine the bending elasticity of DMPC bilayers was increased 30&%slash; -100&%slash; in L&_slash;alpha phase. The NSE data confirmed that fluid crystal/ripple gel transition temperature of DMPC bilayers was depressed by the addition of local anesthetics, which has also been examined via differential scanning calorimetry (DSC).

  6. Dough, tough, cough, rough: A "fast" fMRI localizer of component processes in reading.

    PubMed

    Malins, Jeffrey G; Gumkowski, Nina; Buis, Bonnie; Molfese, Peter; Rueckl, Jay G; Frost, Stephen J; Pugh, Kenneth R; Morris, Robin; Mencl, W Einar

    2016-10-01

    In the current study, we present a novel fMRI protocol in which words, pseudowords, and other word-like stimuli are passively presented in a rapid, sequential fashion. In this "fast" localizer paradigm, items are presented in groups of four; within sets, words are related in orthographic, phonological, and/or semantic properties. We tested this protocol with a group of skilled adult readers (N=18). Analyses uncovered key regions of the reading network that were sensitive to different component processes at the group level; namely, left fusiform gyrus as well as the pars opercularis subregion of inferior frontal gyrus were sensitive to lexicality; several regions including left precentral gyrus and left supramarginal gyrus were sensitive to spelling-sound consistency; the pars triangularis subregion of inferior frontal gyrus was sensitive to semantic similarity. Additionally, in a number of key brain regions, activation in response to semantically similar words was related to individual differences in reading comprehension outside the scanner. Importantly, these findings are in line with previous investigations of the reading network, yet data were obtained using much less imaging time than comparable paradigms currently available, especially relative to the number of indices of component processes obtained. This feature, combined with the relatively simple nature of the task, renders it appropriate for groups of subjects with a wide range of reading abilities, including children with impairments.

  7. High Accuracy Passive Magnetic Field-Based Localization for Feedback Control Using Principal Component Analysis.

    PubMed

    Foong, Shaohui; Sun, Zhenglong

    2016-08-12

    In this paper, a novel magnetic field-based sensing system employing statistically optimized concurrent multiple sensor outputs for precise field-position association and localization is presented. This method capitalizes on the independence between simultaneous spatial field measurements at multiple locations to induce unique correspondences between field and position. This single-source-multi-sensor configuration is able to achieve accurate and precise localization and tracking of translational motion without contact over large travel distances for feedback control. Principal component analysis (PCA) is used as a pseudo-linear filter to optimally reduce the dimensions of the multi-sensor output space for computationally efficient field-position mapping with artificial neural networks (ANNs). Numerical simulations are employed to investigate the effects of geometric parameters and Gaussian noise corruption on PCA assisted ANN mapping performance. Using a 9-sensor network, the sensing accuracy and closed-loop tracking performance of the proposed optimal field-based sensing system is experimentally evaluated on a linear actuator with a significantly more expensive optical encoder as a comparison.

  8. High Accuracy Passive Magnetic Field-Based Localization for Feedback Control Using Principal Component Analysis

    PubMed Central

    Foong, Shaohui; Sun, Zhenglong

    2016-01-01

    In this paper, a novel magnetic field-based sensing system employing statistically optimized concurrent multiple sensor outputs for precise field-position association and localization is presented. This method capitalizes on the independence between simultaneous spatial field measurements at multiple locations to induce unique correspondences between field and position. This single-source-multi-sensor configuration is able to achieve accurate and precise localization and tracking of translational motion without contact over large travel distances for feedback control. Principal component analysis (PCA) is used as a pseudo-linear filter to optimally reduce the dimensions of the multi-sensor output space for computationally efficient field-position mapping with artificial neural networks (ANNs). Numerical simulations are employed to investigate the effects of geometric parameters and Gaussian noise corruption on PCA assisted ANN mapping performance. Using a 9-sensor network, the sensing accuracy and closed-loop tracking performance of the proposed optimal field-based sensing system is experimentally evaluated on a linear actuator with a significantly more expensive optical encoder as a comparison. PMID:27529253

  9. Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis

    PubMed Central

    1989-01-01

    A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn- 78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with

  10. The Investigation and Development of Low Cost Hardware Components for Proton-Exchange Membrane Fuel Cells - Final Report

    SciTech Connect

    George A. Marchetti

    1999-12-15

    Proton exchange membrane (PEM) fuel cell components, which would have a low-cost structure in mass production, were fabricated and tested. A fuel cell electrode structure, comprising a thin layer of graphite (50 microns) and a front-loaded platinum catalyst layer (600 angstroms), was shown to produce significant power densities. In addition, a PEM bipolar plate, comprising flexible graphite, carbon cloth flow-fields and an integrated polymer gasket, was fabricated. Power densities of a two-cell unit using this inexpensive bipolar plate architecture were shown to be comparable to state-of-the-art bipolar plates.

  11. F plasmid TraF and TraH are components of an outer membrane complex involved in conjugation.

    PubMed

    Arutyunov, Denis; Arenson, Barbara; Manchak, Jan; Frost, Laura S

    2010-03-01

    F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.

  12. F Plasmid TraF and TraH Are Components of an Outer Membrane Complex Involved in Conjugation▿

    PubMed Central

    Arutyunov, Denis; Arenson, Barbara; Manchak, Jan; Frost, Laura S.

    2010-01-01

    F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH. PMID:20081027

  13. Identification of a Zn(2+)-sensitive component of Ehrlich cell plasma membrane redox system by CHAPS-agarose-polyacrylamide electrophoresis and in situ staining of activity.

    PubMed

    Rodríguez-Caso, L; Rodríguez-Agudo, D; del Castillo-Olivares, A; Márquez, J; Núñez de Castro, I; Medina, M A

    1997-01-01

    A procedure based on CHAPS-agarose-polyacrylamide electrophoresis and in situ staining of activity was used to detect a Zn(2+)-sensitive component of Ehrlich cell plasma membrane redox system. The procedure is so powerful that it allows to use crude plasma membrane fractions and can be easily adapted for use in an electrophoretic approach to the purification of this protein.

  14. Localization of core lipopolysaccharide on the inner membrane of Salmonella typhimurium

    SciTech Connect

    Marino, P.A.; Osborn, M.J.

    1986-05-01

    The mechanism of lipopolysaccharide (LPS) translocation to the outer membrane (OM) is poorly defined. The authors have used affinity purified antiR/sub a/ LPS-IgG in immunoelectron microscopy and /sup 125/I protein A radio-labeling experiments to localize newly synthesized R/sub a/ LPS on the inner and outer faces of the inner membrane (IM)/sup a/. Cells were pulsed with galactose for 5 min to permit synthesis of R/sub a/ LPS and chased 0-20' by dilution into glucose medium. LPS translocation was halted by addition of DNP at 0/sup 0/. Samples +/-gal were spheroplasted or french pressed to expose outer and inner faces of the IM respectively. Immunoelectron microscopy employed ferritin conjugated goat anti-rabbit IgG as secondary Ab. Galactose pulsed cells showed no apparent labeling of core-LPS on the outer face of the IM. The outer face of the OM was well decorated. French pressed IM vesicles were purified by isopycnic sucrose gradient centrifugation and incubated sequentially with anti-R/sub a/ LPS and /sup 125/I protein A. /sup 125/I labeling was increased 5-fold over gal-negative controls, consistent with exposure of R/sub a/ LPS on the inner face of the IM. IM isolated after treatment of intact spheroplasts with anti-R/sub a/ LPS and /sup 125/I protein A showed no /sup 125/I labeling, although good radiolabeling of OM was found in all cases. These results suggest that core LPS is synthesized on the inner face of the IM and does not accumulate on the outer face in amounts detectable by these methods before translocation to the OM.

  15. Regulation of photosynthetic membrane components in cyanobacteria. Annual report, June 1, 1991 to May 31, 1992

    SciTech Connect

    Sherman, L.A.

    1992-12-31

    The goals of this proposal were two-fold: (1) to analyze the impact of mutations in the Mn-stabilizing protein (MSP) on O{sub 2}-evolution; and (2) to analyze the effect of iron deficiency on membrane assembly in cyanobacteria. The mutations in the psbO gene were performed in the transformable and photoheterotrophic cyanobacterium Synechococcus sp. PCC6803; this strain allows PSII mutations to be propagated under nonphotosynthetic conditions. The research with iron deficiency was performed in the cyanobacterium Synechococcus sp. PCC7942, which is transformable and which has been used previously for all of the authors nutritional-deficiency research. The Synechocystis psbO gene encodes the 33 kDa Mn-stabilizing protein (MSP) of PSII. MSP is an extrinsic protein situated on the lumenal face on the thylakoid membrane, and has been implicated in the stabilization of two of the four Mn atoms, which form the catalytic center of the H{sub 2}O-splitting reaction. There has been a long-standing controversy surrounding the problem. All previous genetic results indicated that MSP was required for O{sub 2}-evolution in vivo. In contrast, biochemical depletion/reconstitution studies suggested that MSP is not absolutely essential, but promotes optimal rates of O{sub 2}-evolving activity by accelerating certain catalytic steps of the reaction cycle.

  16. Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus.

    PubMed

    Jäger, Franziska; Zoltner, Martin; Kneuper, Holger; Hunter, William N; Palmer, Tracy

    2016-02-01

    The Ess/Type VII protein secretion system, essential for virulence of pathogenic Staphylococcus aureus, is dependent upon the four core membrane proteins EssA, EssB, EssC and EsaA. Here, we use crosslinking and blue native PAGE analysis to show that the EssB, EssC and EsaA proteins individually form homomeric complexes. Surprisingly, these components appear unable to interact with each other, or with the EssA protein. We further show that two high molecular weight multimers of EssC detected in whole cells are not dependent upon the presence of EsxA, EsxB or any other Ess component for their assembly. © 2016 Federation of European Biochemical Societies.

  17. Staphylococcus aureus Hemolysins, bi-component Leukocidins, and Cytolytic Peptides: A Redundant Arsenal of Membrane-Damaging Virulence Factors?

    PubMed Central

    Vandenesch, François; Lina, G.; Henry, Thomas

    2012-01-01

    One key aspect of the virulence of Staphylococcus aureus lies in its ability to target the host cell membrane with a large number of membrane-damaging toxins and peptides. In this review, we describe the hemolysins, the bi-component leukocidins (which include the Panton Valentine leukocidin, LukAB/GH, and LukED), and the cytolytic peptides (phenol soluble modulins). While at first glance, all of these factors might appear redundant, it is now clear that some of these factors play specific roles in certain S. aureus life stages and diseases or target specific cell types or species. In this review, we present an update of the literature on toxin receptors and their cell type and species specificities. Furthermore, we review epidemiological studies and animal models illustrating the role of these membrane-damaging factors in various diseases. Finally, we emphasize the interplay of these factors with the host immune system and highlight all their non-lytic functions. PMID:22919604

  18. Linking Findings in Microfluidics to Membrane Emulsification Process Design: The Importance of Wettability and Component Interactions with Interfaces

    PubMed Central

    Schroën, Karin; Ferrando, Montse; de Lamo-Castellví, Silvia; Sahin, Sami; Güell, Carme

    2016-01-01

    In microfluidics and other microstructured devices, wettability changes, as a result of component interactions with the solid wall, can have dramatic effects. In emulsion separation and emulsification applications, the desired behavior can even be completely lost. Wettability changes also occur in one phase systems, but the effect is much more far-reaching when using two-phase systems. For microfluidic emulsification devices, this can be elegantly demonstrated and quantified for EDGE (Edge-base Droplet GEneration) devices that have a specific behavior that allows us to distinguish between surfactant and liquid interactions with the solid surface. Based on these findings, design rules can be defined for emulsification with any micro-structured emulsification device, such as direct and premix membrane emulsification. In general, it can be concluded that mostly surface interactions increase the contact angle toward 90°, either through the surfactant, or the oil that is used. This leads to poor process stability, and very limited pressure ranges at which small droplets can be made in microfluidic systems, and cross-flow membrane emulsification. In a limited number of cases, surface interactions can also lead to lower contact angles, thereby increasing the operational stability. This paper concludes with a guideline that can be used to come to the appropriate combination of membrane construction material (or any micro-structured device), surfactants and liquids, in combination with process conditions. PMID:27187484

  19. A Potential Nanofiber Membrane Device for Filling Surgical Residual Cavity to Prevent Glioma Recurrence and Improve Local Neural Tissue Reconstruction

    PubMed Central

    Huang, Daoxiang; Lin, Chao; Wen, Xuejun; Gu, Shuying; Zhao, Peng

    2016-01-01

    This study aims to develop a novel device with nanofiber membrane capable of sustained release of temozolomide (TMZ) and neuron growth factor (NGF). An improved bio-availability of TMZ and NGF in surroundings proximal to the device was expected to be attained for a prolonged period of time. The device was developed by integrating TMZ-doped polycaprolactone (PCL) nanofiber (TP) membrane and NGF-coated PCL (NGFP) membrane using sodium alginate hydrogel. TP was prepared by direct electrospinning of TMZ/PCL. NGFP membrane was developed by layer-by-layer assembling technology. The incorporation of TMZ-doped nanofiber and NGFP nanofiber in the device was confirmed by scanning electron microscopy. The number of NGF layer in NGF-coated PCL membrane could be readily measured with energy spectrum analysis. The in vitro release study showed that TP-NGFP-TP membrane could efficiently liberate TMZ to inhibit the growth of C6 glioma cells, and sufficient NGF to induce the differentiation of PC12 neuron cells over four weeks. Such TP-NGFP-TP membrane device can be employed as a tampon to fill up surgical residual cavity and afford residual glioma removal, structural support, hemostasis, and local neural tissue reconstruction in the surgical treatment of glioma. The study opens a horizon to develop multifunctional biomaterial device for maximized glioma treatment efficacy. PMID:27548322

  20. A Potential Nanofiber Membrane Device for Filling Surgical Residual Cavity to Prevent Glioma Recurrence and Improve Local Neural Tissue Reconstruction.

    PubMed

    Huang, Daoxiang; Lin, Chao; Wen, Xuejun; Gu, Shuying; Zhao, Peng

    2016-01-01

    This study aims to develop a novel device with nanofiber membrane capable of sustained release of temozolomide (TMZ) and neuron growth factor (NGF). An improved bio-availability of TMZ and NGF in surroundings proximal to the device was expected to be attained for a prolonged period of time. The device was developed by integrating TMZ-doped polycaprolactone (PCL) nanofiber (TP) membrane and NGF-coated PCL (NGFP) membrane using sodium alginate hydrogel. TP was prepared by direct electrospinning of TMZ/PCL. NGFP membrane was developed by layer-by-layer assembling technology. The incorporation of TMZ-doped nanofiber and NGFP nanofiber in the device was confirmed by scanning electron microscopy. The number of NGF layer in NGF-coated PCL membrane could be readily measured with energy spectrum analysis. The in vitro release study showed that TP-NGFP-TP membrane could efficiently liberate TMZ to inhibit the growth of C6 glioma cells, and sufficient NGF to induce the differentiation of PC12 neuron cells over four weeks. Such TP-NGFP-TP membrane device can be employed as a tampon to fill up surgical residual cavity and afford residual glioma removal, structural support, hemostasis, and local neural tissue reconstruction in the surgical treatment of glioma. The study opens a horizon to develop multifunctional biomaterial device for maximized glioma treatment efficacy.

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

    PubMed

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

    2010-12-10

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

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

    SciTech Connect

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

    2010-12-10

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

  3. The Role of Progesterone and a Novel Progesterone Receptor, Progesterone Receptor Membrane Component 1, in the Inflammatory Response of Fetal Membranes to Ureaplasma parvum Infection.

    PubMed

    Feng, Liping; Ransom, Carla E; Nazzal, Matthew K; Allen, Terrence K; Li, Yi-Ju; Truong, Tracy; Potts, Lauren C; Seed, Patrick C; Murtha, Amy P

    2016-01-01

    Ureaplasma parvum (U. parvum) is gaining recognition as an important pathogen for chorioamnionitis and preterm premature rupture of membranes. We aimed to investigate the roles of progesterone (P4) and a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), in the response of fetal membranes to U. parvum. Fetal membrane cells (amnion, chorion and decidua) were isolated and confirmed to be free of Mycoplasmataceae. Cells were treated with U. parvum (5x106 CFU), and adherence was quantified by qPCR. Amnion and chorion cells were transfected with scrambled siRNA or validated PGRMC1 siRNA for 72h. Cells were then treated with U. parvum for 4h with or without pretreatment with P4 (10-7 M) or ethanol for 1h. Interleukin-8 (IL-8), matrix metalloproteinase 9 (MMP9) and cyclooxygenase (COX-2) mRNA expression were quantified by qRT-PCR. Culture medium was harvested and analyzed for IL-8 and prostaglandin (PGE2) secretion by ELISA and MMP9 activity by zymography. U. parvum had a mean adherence of 15.0±0.6%, 16.9± 3.7% and 4.7±0.3% in cultured amnion, chorion and decidua cells, respectively. Exposure to U. parvum elicited significant inflammatory responses including induction of IL-8, COX-2, PGE2 and MMP9. A possible role of PGRMC1 was identified in the inhibition of U. parvum-stimulated COX-2 and MMP9 mRNA expression in chorion cells and MMP9 activity in amnion cells. On the other hand, it might enhance the U. parvum-stimulated IL-8 protein secretion in amnion cells. P4, mediated through PGRMC1, significantly inhibited U. Parvum-induced MMP9 mRNA and COX-2 mRNA expression in chorion cells. P4 appeared to attenuate U. parvum induced IL-8 mRNA expression in chorion cells, but this P4 effect might not mediated through PGRMC1. In summary, U. parvum preferentially adheres to and induces inflammatory responses in chorion and amnion cells. P4 and PGRMC1 appear to differentially modulate the inflammatory responses induced by U. parvum among amnion and

  4. The Role of Progesterone and a Novel Progesterone Receptor, Progesterone Receptor Membrane Component 1, in the Inflammatory Response of Fetal Membranes to Ureaplasma parvum Infection

    PubMed Central

    Feng, Liping; Ransom, Carla E.; Nazzal, Matthew K.; Allen, Terrence K.; Li, Yi-Ju; Truong, Tracy; Potts, Lauren C.; Seed, Patrick C.; Murtha, Amy P.

    2016-01-01

    Ureaplasma parvum (U. parvum) is gaining recognition as an important pathogen for chorioamnionitis and preterm premature rupture of membranes. We aimed to investigate the roles of progesterone (P4) and a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), in the response of fetal membranes to U. parvum. Fetal membrane cells (amnion, chorion and decidua) were isolated and confirmed to be free of Mycoplasmataceae. Cells were treated with U. parvum (5x106 CFU), and adherence was quantified by qPCR. Amnion and chorion cells were transfected with scrambled siRNA or validated PGRMC1 siRNA for 72h. Cells were then treated with U. parvum for 4h with or without pretreatment with P4 (10−7 M) or ethanol for 1h. Interleukin-8 (IL-8), matrix metalloproteinase 9 (MMP9) and cyclooxygenase (COX-2) mRNA expression were quantified by qRT-PCR. Culture medium was harvested and analyzed for IL-8 and prostaglandin (PGE2) secretion by ELISA and MMP9 activity by zymography. U. parvum had a mean adherence of 15.0±0.6%, 16.9± 3.7% and 4.7±0.3% in cultured amnion, chorion and decidua cells, respectively. Exposure to U. parvum elicited significant inflammatory responses including induction of IL-8, COX-2, PGE2 and MMP9. A possible role of PGRMC1 was identified in the inhibition of U. parvum-stimulated COX-2 and MMP9 mRNA expression in chorion cells and MMP9 activity in amnion cells. On the other hand, it might enhance the U. parvum-stimulated IL-8 protein secretion in amnion cells. P4, mediated through PGRMC1, significantly inhibited U. Parvum-induced MMP9 mRNA and COX-2 mRNA expression in chorion cells. P4 appeared to attenuate U. parvum induced IL-8 mRNA expression in chorion cells, but this P4 effect might not mediated through PGRMC1. In summary, U. parvum preferentially adheres to and induces inflammatory responses in chorion and amnion cells. P4 and PGRMC1 appear to differentially modulate the inflammatory responses induced by U. parvum among amnion and

  5. A Novel Role for Progesterone Receptor Membrane Component 1 (PGRMC1): A Partner and Regulator of Ferrochelatase.

    PubMed

    Piel, Robert B; Shiferaw, Mesafint T; Vashisht, Ajay A; Marcero, Jason R; Praissman, Jeremy L; Phillips, John D; Wohlschlegel, James A; Medlock, Amy E

    2016-09-20

    Heme is an iron-containing cofactor essential for multiple cellular processes and fundamental activities such as oxygen transport. To better understand the means by which heme synthesis is regulated during erythropoiesis, affinity purification coupled with mass spectrometry (MS) was performed to identify putative protein partners interacting with ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Both progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) were identified in these experiments. These interactions were validated by reciprocal affinity purification followed by MS analysis and immunoblotting. The interaction between PGRMC1 and FECH was confirmed in vitro and in HEK 293T cells, a non-erythroid cell line. When cells that are recognized models for erythroid differentiation were treated with a small molecule inhibitor of PGRMC1, AG-205, there was an observed decrease in the level of hemoglobinization relative to that of untreated cells. In vitro heme transfer experiments showed that purified PGRMC1 was able to donate heme to apo-cytochrome b5. In the presence of PGRMC1, in vitro measured FECH activity decreased in a dose-dependent manner. Interactions between FECH and PGRMC1 were strongest for the conformation of FECH associated with product release, suggesting that PGRMC1 may regulate FECH activity by controlling heme release. Overall, the data illustrate a role for PGRMC1 in regulating heme synthesis via interactions with FECH and suggest that PGRMC1 may be a heme chaperone or sensor.

  6. Tom40, the Pore-Forming Component of the Protein-Conducting Tom Channel in the Outer Membrane of Mitochondria

    PubMed Central

    Ahting, Uwe; Thieffry, Michel; Engelhardt, Harald; Hegerl, Reiner; Neupert, Walter; Nussberger, Stephan

    2001-01-01

    Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of ∼350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises ∼31% β-sheet, 22% α-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of ∼2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly. PMID:11402060

  7. Palmitoylation of the Immunity Related GTPase, Irgm1: Impact on Membrane Localization and Ability to Promote Mitochondrial Fission

    PubMed Central

    Henry, Stanley C.; Schmidt, Elyse A.; Fessler, Michael B.; Taylor, Gregory A.

    2014-01-01

    The Immunity-Related GTPases (IRG) are a family of large GTPases that mediate innate immune responses. Irgm1 is particularly critical for immunity to bacteria and protozoa, and for inflammatory homeostasis in the intestine. Although precise functions for Irgm1 have not been identified, prior studies have suggested roles in autophagy/mitophagy, phagosome remodeling, cell motility, and regulating the activity of other IRG proteins. These functions ostensibly hinge on the ability of Irgm1 to localize to intracellular membranes, such as those of the Golgi apparatus and mitochondria. Previously, it has been shown that an amphipathic helix, the αK helix, in the C-terminal portion of the protein partially mediates membrane binding. However, in absence of αK, there is still substantial binding of Irgm1 to cellular membranes, suggesting the presence of other membrane binding motifs. In the current work, an additional membrane localization motif was found in the form of palmitoylation at a cluster of cysteines near the αK. An Irgm1 mutant possessing alanine to cysteine substitutions at these amino acids demonstrated little residual palmitoylation, yet it displayed only a small decrease in localization to the Golgi and mitochondria. In contrast, a mutant containing the palmitoylation mutations in combination with mutations disrupting the amphipathic character of the αK displayed a complete loss of apparent localization to the Golgi and mitochondria, as well as an overall loss of association with cellular membranes in general. Additionally, Irgm1 was found to promote mitochondrial fission, and this function was undermined in Irgm1 mutants lacking the palmitoylation domain, and to a greater extent in those lacking the αK, or the αK and palmitoylation domains combined. Our data suggest that palmitoylation together with the αK helix firmly anchor Irgm1 in the Golgi and mitochondria, thus facilitating function of the protein. PMID:24751652

  8. On the study of local-stress rearrangements during quasi-static plastic shear of a model glass: do local-stress components contain enough information?

    PubMed

    Tsamados, M; Tanguy, A; Léonforte, F; Barrat, J-L

    2008-07-01

    We present a numerical study of the mechanical response of a 2D Lennard-Jones amorphous solid under steady quasi-static and athermal shear. We focus here on the evolution of local stress components. While the local stress is usually taken as an order parameter in the description of the rheological behaviour of complex fluids, and for plasticity in glasses, we show here that the knowledge of local stresses is not sufficient for a complete description of the plastic behaviour of our system. The distribution of local stresses can be approximately described as resulting from the sum of localized quadrupolar events with an exponential distribution of amplitudes. However, we show that the position of the center of the quadrupoles is not related to any special evolution of the local stress, but must be described by another variable.

  9. Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

    PubMed

    Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

    2014-01-01

    Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

  10. Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane

    PubMed Central

    Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H.; Sosinsky, Gina E.

    2015-01-01

    Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system

  11. Complementary Constrains on Component based Multiphase Flow Problems, Should It Be Implemented Locally or Globally?

    NASA Astrophysics Data System (ADS)

    Shao, H.; Huang, Y.; Kolditz, O.

    2015-12-01

    Multiphase flow problems are numerically difficult to solve, as it often contains nonlinear Phase transition phenomena A conventional technique is to introduce the complementarity constraints where fluid properties such as liquid saturations are confined within a physically reasonable range. Based on such constraints, the mathematical model can be reformulated into a system of nonlinear partial differential equations coupled with variational inequalities. They can be then numerically handled by optimization algorithms. In this work, two different approaches utilizing the complementarity constraints based on persistent primary variables formulation[4] are implemented and investigated. The first approach proposed by Marchand et.al[1] is using "local complementary constraints", i.e. coupling the constraints with the local constitutive equations. The second approach[2],[3] , namely the "global complementary constrains", applies the constraints globally with the mass conservation equation. We will discuss how these two approaches are applied to solve non-isothermal componential multiphase flow problem with the phase change phenomenon. Several benchmarks will be presented for investigating the overall numerical performance of different approaches. The advantages and disadvantages of different models will also be concluded. References[1] E.Marchand, T.Mueller and P.Knabner. Fully coupled generalized hybrid-mixed finite element approximation of two-phase two-component flow in porous media. Part I: formulation and properties of the mathematical model, Computational Geosciences 17(2): 431-442, (2013). [2] A. Lauser, C. Hager, R. Helmig, B. Wohlmuth. A new approach for phase transitions in miscible multi-phase flow in porous media. Water Resour., 34,(2011), 957-966. [3] J. Jaffré, and A. Sboui. Henry's Law and Gas Phase Disappearance. Transp. Porous Media. 82, (2010), 521-526. [4] A. Bourgeat, M. Jurak and F. Smaï. Two-phase partially miscible flow and transport modeling in

  12. Tespa1 is a novel component of mitochondria-associated endoplasmic reticulum membranes and affects mitochondrial calcium flux.

    PubMed

    Matsuzaki, Hiroshi; Fujimoto, Takahiro; Tanaka, Masatoshi; Shirasawa, Senji

    2013-04-12

    Regulation of intracellular Ca(2+) concentration is critical in numerous biological processes. Inositol 1,4,5-trisphosphate receptor (IP3R) functions as the Ca(2+) release channel on endoplasmic reticulum (ER) membranes. Much attention has been dedicated to mitochondrial Ca(2+) uptake via mitochondria-associated ER membranes (MAM) which is involved in intracellular Ca(2+) homeostasis; however, the molecular mechanisms that link the MAM to mitochondria still remain elusive. We previously reported that Tespa1 (thymocyte-expressed, positive selection-associated gene 1) expressed in lymphocytes physically interacts with IP3R. In this study, we first performed double-immunocytochemical staining of Tespa1 with a mitochondrial marker or an ER marker on an acute T lymphoblastic leukemia cell line, Jurkat cells, by using anti-ATP synthase or anti-calnexin antibody, respectively, and demonstrated that Tespa1 was localized very close to mitochondria and the Tespa1 localization was overlapped with restricted portion of ER. Next, we examined the effects of Tespa1 on the T cell receptor (TCR) stimulation-induced Ca(2+) flux by using Ca(2+) imaging in Jurkat cells. Reduction of Tespa1 protein by Tespa1-specific siRNA diminished TCR stimulation-induced Ca(2+) flux into both mitochondria and cytoplasm through the analyses of the mitochondrial Ca(2+) indicator (Rhod-2) and the cytoplasmic Ca(2+) indicator (Fluo-4), respectively. Furthermore, co-immunoprecipitation assay in HEK293 cells revealed that exogenous Tespa1 protein physically interacted with a MAM-associated protein, GRP75 (glucose-regulated protein 75), but not with an outer mitochondrial membrane protein, VDAC1 (voltage-dependent anion channel 1). All these results suggested that Tespa1 will participate in the molecular link between IP3R-mediated Ca(2+) release and mitochondrial Ca(2+) uptake in the MAM compartment.

  13. Cholesterol modulates the cellular localization of Orai1 channels and its disposition among membrane domains.

    PubMed

    Bohórquez-Hernández, A; Gratton, Enrico; Pacheco, Jonathan; Asanov, Alexander; Vaca, Luis

    2017-09-13

    Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE. In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern (unobstructed diffusion) opposite to basal cholesterol conditions were most of Orai1 channels moves in a confined space, as assessed by Fluorescence Correlation Spectroscopy, Cav1 overexpression inhibited these alterations maintaining Orai1 into a confined and partially confined movement. These results not only highlight the complex effect of cholesterol regulation on SOCE, but also indicate a direct regulatory effect on Orai1 localization and compartmentalization by this lipid. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Bacterial outer membrane vesicles mediate cytosolic localization of LPS and caspase-11 activation

    PubMed Central

    Vanaja, Sivapriya Kailasan; Russo, Ashley J.; Behl, Bharat; Banerjee, Ishita; Yankova, Maya; Deshmukh, Sachin D.; Rathinam, Vijay A.K.

    2016-01-01

    SUMMARY Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11 however are not cytosolic and the mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here we identify outer membrane vesicles (OMV) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMV are internalized via endocytosis, and LPS is released into the cytosol from early endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMV, reveal the importance of OMV in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMV in enabling the cytosolic entry of LPS and consequently caspase-11 activation during Gram-negative bacterial infections. PMID:27156449

  15. Ligand-dependent localization and function of ORP-VAP complexes at membrane contact sites.

    PubMed

    Weber-Boyvat, Marion; Kentala, Henriikka; Peränen, Johan; Olkkonen, Vesa M

    2015-05-01

    Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP-VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP-VAP association, alters the subcellular targeting of ORP-VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP-VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER-lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP-VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.

  16. Multiphoton-generated localized electron plasma for membrane permeability modification in single cells

    NASA Astrophysics Data System (ADS)

    Merritt, T.; Leblanc, M.; McMillan, J.; Westwood, J.; Khodaparast, G. A.

    2014-03-01

    Successful incorporation of a specific macromolecule into a single cell would be ideal for characterizing trafficking dynamics through plasmodesmata or for studying intracellular localizations. Here, we demonstrate NIR femtosecond laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into living cells of Arabidopsis thaliana seedling stems. Based on the reactions of fluorescing vacuoles of transgenic cells and artificial cell walls comprised of nanocellulose, laser intensity and exposure time were adjusted to avoid deleterious effects. Using these plant-tailored laser parameters, cells were injected with the fluorophores and long-term dye retention was observed, all while preserving vital cell functions. This method is ideal for studies concerning cell-to-cell interactions and potentially paves the way for introducing transgenes to specific cells. This work was supported by NSF award IOS-0843372 to JHW, with additional support from and U.S. Department of Agriculture Hatch Project no. 135997, and by the Institute of Critical Technology and Applied Sciences (ICTAS) at Virginia Tech.

  17. Freeze-Etch Study of Pseudomonas aeruginosa: Localization Within the Cell Wall of an Ethylenediaminetetraacetate-Extractable Component

    PubMed Central

    Gilleland, H. E.; Stinnett, J. D.; Roth, I. L.; Eagon, R. G.

    1973-01-01

    A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg2+, the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system. Images PMID:4120071

  18. Localization of the expression of complement component 3 in the human endometrium by in situ hybridization

    SciTech Connect

    Sayegh, R.A.; Tao, Xiao Jing; Awwad, J.T.

    1996-04-01

    C3 production by the human endometrium has been previously described. The objective of the current study was to localize the site of expression and regulation of the third component of complement, C3, in the endometrium. Eight secretory and eight proliferative archival endometrial samples from hysterectomy and endometrial biopsy specimens were used for in situ hybridization analysis. This analysis was performed with a radiolabeled riboprobe synthesized from a 736-bp template representing sequence 1944-2680 of the human C3 complementary DNA. Duplicate sections were hybridized with sense and antisense riboprobes. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy. In proliferative endometrium, minimal expression of C3 was observed and was limited to a few stromal patches and glands throughout the section. In the secretory samples, prominent C3 expression was observed in both the glands and stroma of the basalis layer. Endometrial lymphocytes did not express C3. Endometrial stromal and glandular cells express the C3 gene. Endometrial lymphocytes did not express C3, but other nondistinct lymphoid elements scattered in the stroma may be expressing C3. There was a visibly more intense expression of C3 in the basalis layer of the secretory endometrium than in proliferative endometrium. The spatial and temporal pattern of C3 expression may have implications in normal menstrual physiology and in the immunological response of the endometrium to the invading trophoblast during placentation. 23 refs., 4 figs., 1 tab.

  19. High spectral specificity of local chemical components characterization with multichannel shift-excitation Raman spectroscopy

    PubMed Central

    Chen, Kun; Wu, Tao; Wei, Haoyun; Wu, Xuejian; Li, Yan

    2015-01-01

    Raman spectroscopy has emerged as a promising tool for its noninvasive and nondestructive characterization of local chemical structures. However, spectrally overlapping components prevent the specific identification of hyperfine molecular information of different substances, because of limitations in the spectral resolving power. The challenge is to find a way of preserving scattered photons and retrieving hidden/buried Raman signatures to take full advantage of its chemical specificity. Here, we demonstrate a multichannel acquisition framework based on shift-excitation and slit-modulation, followed by mathematical post-processing, which enables a significant improvement in the spectral specificity of Raman characterization. The present technique, termed shift-excitation blind super-resolution Raman spectroscopy (SEBSR), uses multiple degraded spectra to beat the dispersion-loss trade-off and facilitate high-resolution applications. It overcomes a fundamental problem that has previously plagued high-resolution Raman spectroscopy: fine spectral resolution requires large dispersion, which is accompanied by extreme optical loss. Applicability is demonstrated by the perfect recovery of fine structure of the C-Cl bending mode as well as the clear discrimination of different polymorphs of mannitol. Due to its enhanced discrimination capability, this method offers a feasible route at encouraging a broader range of applications in analytical chemistry, materials and biomedicine. PMID:26350355

  20. Localization of interchromatin granule cluster and Cajal body components in oocyte nuclear bodies of the hemipterans.

    PubMed

    Bogolyubov, D S; Batalova, F M; Ogorzałek, A

    2007-10-01

    An oocyte nucleus contains different extrachromosomal nuclear domains collectively called nuclear bodies (NBs). In the present work we revealed, using immunogold labeling electron microscopy, some marker components of interchromatin granule clusters (IGCs) and Cajal bodies (CBs) in morphologically heterogeneous oocyte NBs studied in three hemipteran species: Notostira elongata, Capsodes gothicus (Miridae) and Velia caprai (Veliidae). Both IGC and CB counterparts were revealed in oocyte nuclei of the studied species but morphological and biochemical criteria were found to be not sufficient to determine carefully the define type of oocyte NBs. We found that the molecular markers of the CBs (coilin and non-phosphorylated RNA polymerase II) and IGCs (SC35 protein) may be localized in the same NB. Anti-SC35 antibody may decorate not only a granular material representing "true" interchromatin granules but also masks some fibrillar parts of complex NBs. Our first observations on the hemipteran oocyte NBs confirm the high complexity and heterogeneity of insect oocyte IGCs and CBs in comparison with those in mammalian somatic cells and amphibian oocytes.

  1. Combining Sprague-Dawley rat uterus cell membrane chromatography with HPLC/MS to screen active components from Leonurus artemisia.

    PubMed

    Fan, Jiangbo; Wei, Fen; Zhang, Yu; Su, Hongli; Ji, Zongzheng; He, Jianyu; Han, Shengli

    2016-01-01

    Leonurus artemisia (Lour.) S.Y.Hu (Lamiaceae) (YiMuCao in Chinese) is a traditional Chinese medicine. Leonurus artemisia has been shown to have many pharmacological effects such as increasing uterine contraction amplitude, and tension, but the active components are still unknown. The objective of this study is to determine active components of L. Artemisia that are responsible for the biological activity using HPLC and cell membrane-based system. The whole L. artemisia ethanol extract and its eight fractions were screened using Sprague-Dawley rat uterus cell membrane chromatography (CMC) combined with the HPLC/MS system. Oxytocin was used to investigate the activity of CMC column. The effect of active components screened from L. artemisia was studied by tension measurement of isolated rat uterine strips in vitro at a dose of 10(-7)-10(-4 )mol/L with oxytocin as a control. The acetone extract showed obvious activity when compared with the eight extracts of L. artemisia. From the acetone extract, in the negative ionization mode, the active compound was identified as genkwanin, with a molecular weight of 283. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions at a dose from 10(-7) to 10(-4 )mol/L. The EC50 value was 4.86 ± 4.21 μmol/L for genkwanin and 4.30 ± 3.65 μmol/L for oxytocin on the contractile amplitude of uterine strips isolated from rats. Genkwanin was identified as the active compound in L. artemisia by this method. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions. Genkwanin may be used to uterine inertia and may have an effect on postpartum hemorrhage.

  2. Interaction of local and general anaesthetics with liposomal membrane models: a QCM-D and DSC study.

    PubMed

    Paiva, José Gabriel; Paradiso, Patrizia; Serro, Ana Paula; Fernandes, Anabela; Saramago, Benilde

    2012-06-15

    The behaviour of four local anaesthetics (lidocaine, levobupivacaine, ropivacaine and tetracaine) and one general anaesthetic (propofol) is compared when interacting with two types of model membranes: supported layers of liposomes and liposomes in solution. Several liposomal compositions were tested: dimyristoylphosphatidylcholine (DMPC), binary mixtures of DMPC with cholesterol (CHOL), and ternary mixtures of dipalmitoylphosphatidylcholine (DPPC), DMPC, and CHOL. A quartz crystal microbalance with dissipation, QCM-D, was used to assess changes in the properties of supported layers of liposomes. The effect of the anaesthetics on the phase behaviour of the liposomes in suspension was determined by differential scanning calorimetry. Both techniques show that all anaesthetics have a fluidizing effect on the model membranes but, apparently, the solid supported liposomes are less affected by the anaesthetics than the liposomes in solution. Although the different anaesthetics were compared at different concentrations, tetracaine and propofol seem to induce the strongest perturbation on the liposome membrane. The resistance of the liposomes to the anaesthetic action was found to increase with the presence of cholesterol, while adding DPPC to the binary mixture DMPC+CHOL does not change its behaviour. The novelty of the present work resides upon three points: (1) the use of supported layers of liposomes as model membranes to study interactions with anaesthetics; (2) application of QCM-D to assess changes of the adsorbed liposomes; (3) a comparison of the effect of local and general anaesthetics interacting with various model membranes in similar experimental conditions.

  3. Iron-regulated expression and membrane localization of the magA protein in Magnetospirillum sp. strain AMB-1.

    PubMed

    Nakamura, C; Kikuchi, T; Burgess, J G; Matsunaga, T

    1995-07-01

    The magA gene from Magnetospirillum sp. strain AMB-1 is required for the synthesis of bacterial magnetic particles (BMPs). This gene has been cloned, sequenced and found to encode a protein which is homologous to the Escherichia coli potassium efflux membrane-binding protein, KefC. By using the firefly luciferase gene (luc) cloned downstream of the magA promoter, the effect of iron on regulation of magA expression was investigated, and transcription of magA was found to be enhanced by low concentrations of iron. Intracellular localization of the MagA protein was studied using magA-luc fusion proteins. The luc gene was cloned downstream of the magA hydrophilic C-terminal domain. Detection of luciferase activity in the cytoplasm, cell membrane, and magnetic particle membrane subcellular fractions confirmed that the MagA fusion protein was localized in the cell membrane. The fusion protein was also detected on the surface of the lipid bilayer covering the magnetic particles. These results suggest that MagA is a membrane-bound protein, the expression of which is enhanced at low iron concentrations.

  4. Toxins in Botanical Dietary Supplements: Blue Cohosh Components Disrupt Cellular Respiration and Mitochondrial Membrane Potential

    PubMed Central

    Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B.; Khan, Ikhlas A.; Nagle, Dale G.; Zhou, Yu-Dong

    2014-01-01

    Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA “Black Box” warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3) exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage. PMID:24328138

  5. Toxins in botanical dietary supplements: blue cohosh components disrupt cellular respiration and mitochondrial membrane potential.

    PubMed

    Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B; Khan, Ikhlas A; Nagle, Dale G; Zhou, Yu-Dong

    2014-01-24

    Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA "black box" warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.

  6. Proton Exchange Membrane fuel cell development with lightweight component materials, phase 1

    NASA Astrophysics Data System (ADS)

    Abens, Sandors

    1995-07-01

    Although the Proton Exchange Membrane (PEM) fuel cell is a leading candidate for an automobile power source through meeting the zero emission requirement, its power density is currently an order of magnitude below the 400 W/kg criterion proposed by the Department of Energy. The major contributors to stack weight are the bipolar gas distribution plates. This effort, performed jointly by Energy Research Corporation (ERC) and Texas A&M University (TAM U), focused on lightweight alternative bipolar plate materials and designs. The electronic conductivity of various candidate materials was evaluated. The emphasis was on conductive plastic materials and porous graphite. Several plastic materials with specific resistance between 0.5 and 0.8 ohm/cm were identified. Preliminary evaluation of lightweight materials was performed in single cell tests. The emphasis was on atmospheric pressure and internally humidified cell operation as a potential means of system simplification and reduction of PEM fuel cell ancillary equipment complexity and weight. The performance of single cells was nearly the same at 1 and 3 atm pressure. At a cell potential of O.6V, a current density of 230 mA/sq cm was reached at 1.7 stoichiometric air flow rate. With lightweight bipolar plates, the DOE power density target may be achieved with unpressurized, internally humidified cell stacks.

  7. Enhanced sludge properties and distribution study of sludge components in electrically-enhanced membrane bioreactor.

    PubMed

    Giwa, Adewale; Ahmed, Iftikhar; Hasan, Shadi Wajih

    2015-08-15

    This study investigated the impact of electric field on the physicochemical and biological characteristics of sludge wasted from an electrically-enhanced membrane bioreactor treating medium-strength raw wastewater. This method offers a chemical-free electrokinetic technique to enhance sludge properties and remove heavy metals. For example, sludge volume index (SVI), time-to-filter (TTF), mean sludge particle diameter (PSD), viscosity, and oxidation-reduction potential (ORP) of 21.7 mL/g, 7 min, 40.2 μm, 3.22 mPa s, and -4.9 mV were reported, respectively. Also, X-ray fluorescence (XRF) and X-ray diffraction (XRD) analyses provided mechanisms for heavy metal removal so as to establish relevant pathways for nutrient recovery. Furthermore, variations in dissolved oxygen (DO), conductivity, viscosity, ORP, total suspended solids (MLSS), and volatile suspended solids (MLVSS) were interrelated to evaluate the quality of wasted sludge. A pathway study on the transport and chemical distribution of nutrients and metals in sludge showed great potential for metal removal and nutrient recovery. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. AMACO is a component of the basement membrane-associated Fraser complex.

    PubMed

    Richardson, Rebecca J; Gebauer, Jan M; Zhang, Jin-Li; Kobbe, Birgit; Keene, Douglas R; Karlsen, Kristina Røkenes; Richetti, Stefânia; Wohl, Alexander P; Sengle, Gerhard; Neiss, Wolfram F; Paulsson, Mats; Hammerschmidt, Matthias; Wagener, Raimund

    2014-05-01

    Fraser syndrome (FS) is a phenotypically variable, autosomal recessive disorder characterized by cryptophthalmus, cutaneous syndactyly, and other malformations resulting from mutations in FRAS1, FREM2, and GRIP1. Transient embryonic epidermal blistering causes the characteristic defects of the disorder. Fras1, Frem1, and Frem2 form the extracellular Fraser complex, which is believed to stabilize the basement membrane. However, several cases of FS could not be attributed to mutations in FRAS1, FREM2, or GRIP1, and FS displays high clinical variability, suggesting that there is an additional genetic, possibly modifying contribution to this disorder. An extracellular matrix protein containing VWA-like domains related to those in matrilins and collagens (AMACO), encoded by the VWA2 gene, has a very similar tissue distribution to the Fraser complex proteins in both mouse and zebrafish. Here, we show that AMACO deposition is lost in Fras1-deficient zebrafish and mice and that Fras1 and AMACO interact directly via their chondroitin sulfate proteoglycan (CSPG) and P2 domains. Knockdown of vwa2, which alone causes no phenotype, enhances the phenotype of hypomorphic Fras1 mutant zebrafish. Together, our data suggest that AMACO represents a member of the Fraser complex.

  9. Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1

    SciTech Connect

    Sakamoto, Hikaru; Sakata, Keiko; Kusumi, Kensuke; Kojima, Mikiko; Sakakibara, Hitoshi; Iba, Koh

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

  10. Photochemical solar energy conversion utilizing semiconductors localized in membrane-mimetic systems

    SciTech Connect

    Fendler, J.H.

    1991-08-31

    Extending the frontiers of colloidal photochemistry and colloidal electrochemistry to solar photochemistry research had been the main objective of this research. More specific objectives of this proposal include the examination of semiconductor-particle-mediated photoelectron transfer and photoelectric effects in different membrane mimetic systems. Emphasis had been placed on developing bilayer lipid membranes and Langmuir-Blodgett films as new membrane-mimetic systems, as well as on the characterization and utilization of these systems.

  11. Localization of lysyl oxidase in hen oviduct: implications in egg shell membrane formation and composition.

    PubMed

    Harris, E D; Blount, J E; Leach, R M

    1980-04-04

    Lysyl oxidase activity was found in the isthmus (the membrane-forming region) of the hen's oviduct in a copper-rich region proximal to the shell gland. Desmosine and isodesmosine, cross-linking compounds associated with mature elastin, were found in hydrolysates of the shell membrane, confirming the necessity for lysyl oxidase in its biosynthesis. Shell membranes from hens fed a copper-deficient diet or a diet supplemented with beta-aminopropionitrile had a reduced content of desmosine and isodesmosine.

  12. Local thermal resonance control of GaInP photonic crystal membrane cavities using ambient gas cooling

    SciTech Connect

    Sokolov, Sergei Lian, Jin; Yüce, Emre; Mosk, Allard P.; Combrié, Sylvain; Lehoucq, Gaelle; De Rossi, Alfredo

    2015-04-27

    We perform spatially dependent tuning of a GaInP photonic crystal cavity using a continuous wave violet laser. Local tuning is obtained by laser heating of the photonic crystal membrane. The cavity resonance shift is measured for different pump positions and for two ambient gases: He and N{sub 2}. We find that the width of the temperature profile induced in the membrane depends strongly on the thermal conductivity of the ambient gas. For He gas, a narrow spatial width of the temperature profile of 2.8 μm is predicted and verified in experiment.

  13. Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

    PubMed Central

    Dyrda, Agnieszka; Cytlak, Urszula; Ciuraszkiewicz, Anna; Lipinska, Agnieszka; Cueff, Anne; Bouyer, Guillaume; Egée, Stéphane; Bennekou, Poul; Lew, Virgilio L.; Thomas, Serge L. Y.

    2010-01-01

    Background The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. Methodology/Principal Findings The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. Conclusions/Significance The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content

  14. Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transduction.

    PubMed

    Mahbub Hasan, A K M; Ou, Zhize; Sakakibara, Keiichi; Hirahara, Shino; Iwasaki, Tetsushi; Sato, Ken-ichi; Fukami, Yasuo

    2007-02-01

    A single-transmembrane protein uroplakin III (UPIII) and its tetraspanin binding-partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol-enriched membrane microdomains or "rafts" and involved in the sperm-egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII-xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm-egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non-ionic detergents (Brij 98 and Triton X-100), chemical cross-linking, co-immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII-xUPIb complex and contributes to the sperm-dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol-dependent membrane microdomains when they were co-expressed, whereas co-expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co-expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm-egg interaction and signaling during Xenopus fertilization.

  15. Components of the plasma membrane of growing axons. III. Saxitoxin binding to sodium channels

    PubMed Central

    1984-01-01

    The density of sodium channels was measured in growing and mature axons of the olfactory nerve of the bullfrog, using as a probe the drug saxitoxin (STX). The toxin binds to control nerves from adult animals in a saturable manner with a dissociation constant of approximately 23 nM at 4 degrees C and a capacity of 72 fmol/mg wet weight, equivalent to about five sites per square micrometer of axolemma. In growing nerves, obtained from adult frogs 4-5 wk following removal of the original nerve, the STX-binding capacity per wet weight of tissue is markedly reduced, to approximately 25% of control values, and appears to decrease in the proximodistal direction. STX-binding data, expressed as STX/mg wet weight, was converted to STX/micron 2 of axolemma using stereologically derived values of membrane area per milligram wet weight of nerve. The axolemmal content (area/mg wet weight) of all regions of growing nerve is substantially decreased compared to controls, but increases in the proximodistal direction by 60%. These changes in axolemmal area result in calculated STX receptor densities (per unit axolemmal area) which, in distal regions, are approximately at the level of the mature nerve and, in proximal regions, are actually increased above controls by 50 to 70%. Upon comparing the axolemmal density of intramembrane particles, reported in the companion paper, with the calculated density of STX receptors in both mature and growing nerves, we find a correlation between STX receptors and intramembrane particles with diameters of 11.5-14.0 nm. The growing axon's gradient of sodium channels and the shift from this gradient to a uniform distribution in the mature axon suggest (a) that sodium channels are inserted into the perikaryal plasmalemma and diffuse from there into the growing axolemma, and (b) that the axolemma undergoes functional maturation during growth. PMID:6325471

  16. Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from Electrophorus electricus electroplax membranes.

    PubMed Central

    Agnew, W S; Levinson, S R; Brabson, J S; Raftery, M A

    1978-01-01

    The tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from electroplax membranes of Electrophorus electricus has been purified. The toxin-binding site could be efficiently solubilized with Lubrol-PX, resulting in an extract of high initial specific activity. Purification was facilitated by the development of a rapid, quantitative binding assay. The binding component was stabilized during purification by the use of mixed lipid/detergent micelles of defined composition, and by the saturation of the site with tetrodotoxin. The purification was achieved by means of a highly selective adsorption of the toxin-binding component to DEASE-Sephadex A-25, followed by desorption at high ionic strength and chromatography over Sepharose 6B. Final peak specific activities were at least 50% of the specific activity expected for a pure, undenatured toxin-binding componenet of 230,000 molecular weight. The purified material exhibited a sedimentation coefficient of approximately 8 S and an unusual Stokes radius of 95 A. Purified material showed a relatively simple pattern on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, being comprised of only three polypeptides. PMID:275831

  17. Clinical trial and local process evaluation of an apheresis system for preparation of white cell-reduced platelet components.

    PubMed

    Adams, M R; Dumont, L J; McCall, M; Heaton, W A

    1998-10-01

    A new method for the consistent preparation of white cell (WBC)-reduced plateletpheresis components, the Spectra Leukoreduction System (LRS), was evaluated by clinical trial and local process validation. The centrifuge-based system was projected to decrease the WBC content of plateletpheresis components to a level below 1 x 10(6) per unit. Phase I and II clinical trials were performed. The manufacturer's claims were then tested at the local level with an ongoing quality assurance program. In Phase I, a cross-over analysis of five subjects compared LRS to standard plateletpheresis procedures in collection efficiency and component quality: a panel of in vitro measures was taken on Day 0 and Day 5. In Phase II, the LRS process was tested on a larger scale (n = 57; control = 58) with component transfusion. Finally, validation, determination of degree of conformance with standards, and ongoing quality control were performed locally on a newly installed instrument. Phase I and II trials revealed no significant differences between LRS and control units in donor or recipient safety and comfort, platelet function and yield, or component volume. WBC per-unit values were significantly different: the LRS median per unit was 3.2 x 10(4) WBCs, versus 81.4 x 10(4) for control units. Assessment of process capability gave an estimate of 99-percent confidence that 99.5 percent of LRS units would be WBC reduced to < 1 x 10(6) WBCs. Local process validation and quality control revealed 90-percent confidence that 99 percent of the units would be WBC reduced and 99.9-percent confidence that 75 percent would exceed platelet yield standards; the process was stable over time. The LRS is safe for apheresis and the component produced is safe for transfusion with platelet function and yield equivalent to controls and WBC reduction superior to controls. Local process evaluation confirmed that component quality meets the goals of the institution.

  18. Studies on the interactions between parabens and lipid membrane components in monolayers at the air/aqueous solution interface.

    PubMed

    Flasiński, Michał; Gawryś, Maciej; Broniatowski, Marcin; Wydro, Paweł

    2016-04-01

    The interactions between parabens (PBs) and lipid components of mammalian and bacterial cell membranes were investigated in model systems of Langmuir monolayers. Me-, Et-, Pr- and Bu-paraben studied in this paper are frequently applied as cosmetics and food preservatives, since they possess broad antimicrobial activity. The mode of PB action is connected with their incorporation into the membrane of bacterial organisms, however; it is not known what is the role of the respective lipid species in this mechanism. This problem is crucial to understand the differences in paraben activity toward individual microorganisms and to shed the light onto the problem of PB cytotoxicity reported in studies on mammalian cells. In this paper, the mentioned aspects were investigated with application of the Langmuir monolayer technique complemented with BAM and GIXD. Our experiments revealed that the influence of PBs depends on their chemical structure, solution concentration and on the class of lipid. The strongest modification of the monolayer characteristics, leading to its collapse at low surface pressure, occurred in the presence of BuPB, having the largest chain. PBs interact preferentially with the monolayers possessing low degree of condensation, whereas for LC state, the effect was weaker and observed only as modification of the 2D unit cells. In the model systems, PBs interact with phospholipids characteristic for mammalian membranes (phosphatidylcholine) stronger than with bacterial (phosphatidylglycerol and cardiolipin). This strong influence of parabens on the model systems composed of animal lipids may explain cytotoxic activity of these preservatives. Copyright © 2016 Elsevier B.V. All rights reserved

  19. Enhancement of Cell Surface Expression and Receptor Functions of Membrane Progestin Receptor α (mPRα) by Progesterone Receptor Membrane Component 1 (PGRMC1): Evidence for a Role of PGRMC1 as an Adaptor Protein for Steroid Receptors

    PubMed Central

    Pang, Yefei; Dong, Jing

    2014-01-01

    A variety of functions have been proposed for progesterone receptor membrane component 1 (PGRMC1), including acting as a component of a membrane progestin receptor and as an adaptor protein. Here we show that stable overexpression of human PGRMC1 in nuclear progesterone receptor (PR)-negative breast cancer cell lines causes increased expression of PGRMC1 and membrane progesterone receptor α (mPRα) on cell membranes that is associated with increased specific [3H]progesterone binding. The membrane progestin binding affinity and specificity were characteristic of mPRα, with a Kd of 4.7 nM and high affinity for the mPR-specific agonist, Org OD 02–0, and low affinity for corticosteroids. Progestin treatment caused activation of G proteins, further evidence for increased expression of functional mPRs on PGRMC1-transfected cell membranes. Immunocytochemical and coimmunoprecipitation studies showed a close association of PGRMC1 with mPRα in cell membranes. Transfection of PGRMC1 into spontaneously immortalized rat granulosa cells was associated with membrane expression of PGRMC1 and mPRα as well as antiapoptotic effects of progestins that were abolished after cotransfection with small interfering RNA for mPRα. These data demonstrate that PGRMC1 can act as an adaptor protein, transporting mPRα to the cell surface, and that the progestin binding and apoptotic functions previously ascribed to PGRMC1 are dependent on cell surface expression of mPRα. Collectively, the results suggest PGRMC1 and mPRα are components of a membrane progesterone receptor protein complex. Increased expression of estrogen receptor β was also observed in the membranes of PGRMC1-transfected cells, suggesting that PGRMC1 can act as an adaptor protein for multiple classes of steroid receptors. PMID:24424068

  20. Local and global components of texture-surround suppression of contour-shape coding.

    PubMed

    Gheorghiu, Elena; Kingdom, Frederick A A

    2012-06-15

    Evidence that contour-shapes and texture-shapes are processed by different mechanisms included the finding that contour-shape aftereffects are reduced when the adaptation stimulus is a texture made of contours rather than a single contour. This phenomenon has been termed texture-surround suppression of contour-shape, or TSSCS. How does TSSCS operate and over what spatial extent? We measured the postadaptation shift in the apparent shape frequency of a single sinusoidal-shaped contour as a function of the number of contours in the adaptor stimulus. Contours were Gabor strings in which the Gabor orientations were either tangential (snakes) or orthogonal (ladders) to the path of the contour. We found that for extended surrounds, the aftereffect was strongly reduced when the surround contours were the same as the central adaptor contour, but not when the Gabors making up the surround contours were opposite-in-orientation to those of the central adaptor. For near surrounds, the aftereffect in a snake contour was unaffected by same-orientation but strongly suppressed by opposite-orientation surrounds, whereas the aftereffect for a ladder-contour was suppressed equally by both same- and opposite-orientation near surrounds. Finally, the strength of surround suppression decreased gradually with increasing spatial separation between center and surround. These results indicate that there are two components to texture-surround suppression in our shape aftereffect: one that is sensitive to opposite-orientation texture surrounds, operates locally, and disrupts contour-processing; the other that is sensitive to same-orientation texture surrounds, is spatially extended, and prevents the shape of the contour from being processed as a contour. We also demonstrate that the observed shape aftereffects are not due to changes in the apparent shape-frequency of the adaptors or the precision with which their shape-frequency is encoded, indicating that TSSCS is not an instance of crowding.

  1. Rice OsVAMP714, a membrane-trafficking protein localized to the chloroplast and vacuolar membrane, is involved in resistance to rice blast disease.

    PubMed

    Sugano, Shoji; Hayashi, Nagao; Kawagoe, Yasushi; Mochizuki, Susumu; Inoue, Haruhiko; Mori, Masaki; Nishizawa, Yoko; Jiang, Chang-Jie; Matsui, Minami; Takatsuji, Hiroshi

    2016-05-01

    Membrane trafficking plays pivotal roles in many cellular processes including plant immunity. Here, we report the characterization of OsVAMP714, an intracellular SNARE protein, focusing on its role in resistance to rice blast disease caused by the fungal pathogen Magnaporthe oryzae. Disease resistance tests using OsVAMP714 knockdown and overexpressing rice plants demonstrated the involvement of OsVAMP714 in blast resistance. The overexpression of OsVAMP7111, whose product is highly homologous to OsVAMP714, did not enhance blast resistance to rice, implying a potential specificity of OsVAMP714 to blast resistance. OsVAMP714 was localized to the chloroplast in mesophyll cells and to the cellular periphery in epidermal cells of transgenic rice plant leaves. We showed that chloroplast localization is critical for the normal OsVAMP714 functioning in blast resistance by analyzing the rice plants overexpressing OsVAMP714 mutants whose products did not localize in the chloroplast. We also found that OsVAMP714 was located in the vacuolar membrane surrounding the invasive hyphae of M. oryzae. Furthermore, we showed that OsVAMP714 overexpression promotes leaf sheath elongation and that the first 19 amino acids, which are highly conserved between animal and plant VAMP7 proteins, are crucial for normal rice plant growths. Our studies imply that the OsVAMP714-mediated trafficking pathway plays an important role in rice blast resistance as well as in the vegetative growth of rice.

  2. A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

    PubMed Central

    Resch, Ulrike; Tsatsaronis, James Anthony; Le Rhun, Anaïs; Stübiger, Gerald; Rohde, Manfred; Kasvandik, Sergo; Holzmeister, Susanne; Tinnefeld, Philip; Wai, Sun Nyunt

    2016-01-01

    ABSTRACT Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. PMID:27803183

  3. Low molecular weight components in an aquatic humic substance as characterized by membrane dialysis and orbitrap mass spectrometry.

    PubMed

    Remucal, Christina K; Cory, Rose M; Sander, Michael; McNeill, Kristopher

    2012-09-04

    Suwannee River fulvic acid (SRFA) was dialyzed through a 100-500 molecular weight cutoff dialysis membrane, and the dialysate and retentate were analyzed by UV-visible absorption and high-resolution Orbitrap mass spectrometry (MS). A significant fraction (36% based on dissolved organic carbon) of SRFA passed through the dialysis membrane. The fraction of SRFA in the dialysate had a different UV-visible absorption spectrum and was enriched in low molecular weight molecules with a more aliphatic composition relative to the initial SRFA solution. Comparison of the SRFA spectra collected by Orbitrap MS and Fourier transform ion cyclotron resonance MS (FT-ICR MS) demonstrated that the mass accuracy of the Orbitrap MS is sufficient for determination of unique molecular formulas of compounds with masses <600 Da in a complex mixture, such as SRFA. The most intense masses detected by Orbitrap MS were found in the 100-200 Da mass range. Many of these low molecular masses corresponded to molecular formulas of previously identified compounds in organic matter, lignin, and plants, and the use of the standard addition method provided an upper concentration estimate of selected target compounds in SRFA. Collectively, these results provide evidence that SRFA contains low molecular weight components that are present individually or in loosely bound assemblies.

  4. Progesterone Receptor Membrane Component 1 Mediates Progesterone-Induced Suppression of Oocyte Meiotic Prophase I and Primordial Folliculogenesis.

    PubMed

    Guo, Meng; Zhang, Cheng; Wang, Yan; Feng, Lizhao; Wang, Zhengpin; Niu, Wanbo; Du, Xiaoyan; Tang, Wang; Li, Yuna; Wang, Chao; Chen, Zhenwen

    2016-11-16

    Well-timed progression of primordial folliculogenesis is essential for mammalian female fertility. Progesterone (P4) inhibits primordial follicle formation under physiological conditions; however, P4 receptor that mediates this effect and its underlying mechanisms are unclear. In this study, we used an in vitro organ culture system to show that progesterone receptor membrane component 1 (PGRMC1) mediated P4-induced inhibition of oocyte meiotic prophase I and primordial follicle formation. We found that membrane-impermeable BSA-conjugated P4 inhibited primordial follicle formation similar to that by P4. Interestingly, PGRMC1 and its partner serpine1 mRNA-binding protein 1 were highly expressed in oocytes in perinatal ovaries. Inhibition or RNA interference of PGRMC1 abolished the suppressive effect of P4 on follicle formation. Furthermore, P4-PGRMC1 interaction blocked oocyte meiotic progression and decreased intra-oocyte cyclic AMP (cAMP) levels in perinatal ovaries. cAMP analog dibutyryl cAMP reversed P4-PGRMC1 interaction-induced inhibition of meiotic progression and follicle formation. Thus, our results indicated that PGRMC1 mediated P4-induced suppression of oocyte meiotic progression and primordial folliculogenesis by decreasing intra-oocyte cAMP levels.

  5. Progesterone Receptor Membrane Component 1 Mediates Progesterone-Induced Suppression of Oocyte Meiotic Prophase I and Primordial Folliculogenesis

    PubMed Central

    Guo, Meng; Zhang, Cheng; Wang, Yan; Feng, Lizhao; Wang, Zhengpin; Niu, Wanbo; Du, Xiaoyan; Tang, Wang; Li, Yuna; Wang, Chao; Chen, Zhenwen

    2016-01-01

    Well-timed progression of primordial folliculogenesis is essential for mammalian female fertility. Progesterone (P4) inhibits primordial follicle formation under physiological conditions; however, P4 receptor that mediates this effect and its underlying mechanisms are unclear. In this study, we used an in vitro organ culture system to show that progesterone receptor membrane component 1 (PGRMC1) mediated P4-induced inhibition of oocyte meiotic prophase I and primordial follicle formation. We found that membrane-impermeable BSA-conjugated P4 inhibited primordial follicle formation similar to that by P4. Interestingly, PGRMC1 and its partner serpine1 mRNA-binding protein 1 were highly expressed in oocytes in perinatal ovaries. Inhibition or RNA interference of PGRMC1 abolished the suppressive effect of P4 on follicle formation. Furthermore, P4-PGRMC1 interaction blocked oocyte meiotic progression and decreased intra-oocyte cyclic AMP (cAMP) levels in perinatal ovaries. cAMP analog dibutyryl cAMP reversed P4–PGRMC1 interaction-induced inhibition of meiotic progression and follicle formation. Thus, our results indicated that PGRMC1 mediated P4-induced suppression of oocyte meiotic progression and primordial folliculogenesis by decreasing intra-oocyte cAMP levels. PMID:27848973

  6. Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

    PubMed Central

    Lieberman, Joshua A.; Frost, Nicholas A.; Hoppert, Michael; Fernandes, Paula J.; Vogt, Stefanie L.; Raivio, Tracy L.; Blanpied, Thomas A.

    2012-01-01

    Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis. PMID:22247509

  7. Antibody response of swine to outer membrane components of Haemophilus pleuropneumoniae during infection.

    PubMed Central

    Rapp, V J; Ross, R F

    1986-01-01

    Sera from pigs infected with Haemophilus (Actinobacillus) pleuropneumoniae were tested for antibodies to outer membrane proteins (OMPs) of the organism by immunoblotting. Convalescent sera were produced in naturally born, colostrum-fed pigs and in cesarean-derived, colostrum-deprived pigs given H. pleuropneumoniae serotype 5 intranasally twice at 5-week intervals. Sera, collected at weekly intervals, were reacted with Sarkosyl-insoluble, OMP-enriched preparations of H. pleuropneumoniae which had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Antibodies were detected to OMPs with an apparent molecular weight of 16,500 (16.5K OMP); to 29K, 38.5K, 43.5K, 45K, 49.5K, and 66.5K OMPs; and to several high-molecular-weight (greater than or equal to 94,000) OMPs, but not to the major 42K OMP. Antibodies to the heat-modifiable OMP (29K/43.5K) and the 38.5K OMP were detected in sera from noninfected pigs. Antibodies were also detected to two broad 54,000- and 95,000-molecular-weight bands which did not stain with Coomassie blue, stained with silver nitrate, resisted proteinase K digestion, and were eliminated by oxidation with sodium metaperiodate. This indicates that the 54,000- and 95,000-molecular-weight bands represent polysaccharide, possibly capsular or lipopolysaccharide immunogens. Adsorption of sera with cells from the homologous serotype 5 strain removed antibodies to the 45K, 49.5K, 66.5K, and greater than or equal to 94K OMPs and to the two polysaccharide bands, indicating that these antibodies were directed primarily to surface-exposed epitopes. When tested with OMP preparations from other serotype 5 strains, heterogeneity was apparent, both in the reactions with OMPs and with the polysaccharide bands. Silver staining of proteinase K-treated, whole-cell lysates from serotype 5 strains also indicated variable expression of the polysaccharide bands. Sera also reacted with OMPs from

  8. Properties and degradation of the gasket component of a proton exchange membrane fuel cell--a review.

    PubMed

    Basuli, Utpal; Jose, Jobin; Lee, Ran Hee; Yoo, Yong Hwan; Jeong, Kwang-Un; Ahn, Jou-Hyeon; Nah, Changwoon

    2012-10-01

    Proton exchange membrane (PEM) fuel cell stack requires gaskets and seals in each cell to keep the reactant gases within their respective regions. Gasket performance is integral to the successful long-term operation of a fuel cell stack. This review focuses on properties, performance and degradation mechanisms of the different polymer gasket materials used in PEM fuel cell under normal operating conditions. The different degradation mechanisms and their corresponding representative mitigation strategies are also presented here. Summary of various properties of elastomers and their advantages and disadvantages in fuel cell'environment are presented. By considering the level of chemical degradation, mechanical properties and cost effectiveness, it can be proposed that EPDM is one of the best choices for gasket material in PEM fuel cell. Finally, the challenges that remain in using rubber component as in PEM fuel cell, as well as the prospects for exploiting them in the future are discussed.

  9. Differential cytotoxicity of MEX: a component of Neem oil whose action is exerted at the cell membrane level.

    PubMed

    Ricci, Francesca; Berardi, Valerio; Risuleo, Gianfranco

    2008-12-31

    Neem oil is obtained from the seeds of the tree Azadirachta indica. Its chemical composition is very complex, being rich in terpenoids and limonoids, as well as volatile sulphur modified compounds. This work focused on the evaluation of a component of the whole Neem oil obtained by methanolic extraction and defined as MEX. Cytotoxicity was assessed on two different cell populations: a stabilized murine fibroblast line (3T6) and a tumor cell line (HeLa). The data presented here suggest a differential sensitivity of these two populations, the tumor line exhibiting a significantly higher sensitivity to MEX. The data strongly suggest that its toxic target is the cell membrane. In addition the results presented here imply that MEX may contain one or more agents that could find a potential use in anti-proliferative therapy.

  10. Mechanisms of use-dependent block of sodium channels in excitable membranes by local anesthetics.

    PubMed Central

    Starmer, C F; Grant, A O; Strauss, H C

    1984-01-01

    Many local anesthetics promote reduction in sodium current during repetitive stimulation of excitable membranes. Use-, frequency-, and voltage-dependent responses describe patterns of peak INa when pulse width, pulse frequency, and pulse amplitude are varied. Such responses can be viewed as reflecting voltage-sensitive shifts in equilibrium between conducting, unblocked channels and nonconducting, blocked channels. The modulated-receptor hypothesis postulates shifts in equilibrium as the result of a variable-affinity receptor and modified inactivation gate kinetics in drug-complexed channels. An alternative view considers drug blocking in the absence of these two features. We propose that drug binds to a constant-affinity channel receptor where receptor access is regulated by the channel gates. Specifically, we view channel binding sites as guarded by the channel gate conformation, so that unlike receptors where ligands have continuous access, blocking agent access is variable during the course of an action potential. During the course of an action potential, the m and h gates change conformation in response to transmembrane potential. Conducting channels with both gates open leave the binding site unguarded and thus accessible to drug, whereas nonconducting channels, with gates in the closed conformation, act to restrict drug access to unbound receptors and possibly to trap drug in drug-complexed channels. We develop analytical expressions characterizing guarded receptors as "apparently" variable-affinity binding sites and predicting shifts in "apparent" channel inactivation in the hyperpolarizing direction. These results were confirmed with computer simulations. Furthermore, these results are in quantitative agreement with recent investigations of lidocaine binding in cardiac sodium channels. PMID:6331543

  11. Bile salt-stimulated phospholipid efflux mediated by ABCB4 localized in nonraft membranes.

    PubMed

    Morita, Shin-ya; Tsuda, Tadanori; Horikami, Manami; Teraoka, Reiko; Kitagawa, Shuji; Terada, Tomohiro

    2013-05-01

    ABCB4 is necessary for the secretion of phospholipids from hepatocytes into bile and for the protection of cell membranes against bile salts. Lipid rafts are plasma membrane microdomains containing high contents of cholesterol and sphingolipids, which are separated by Triton X-100 extraction or OptiPrep gradient centrifugation. In this study, we investigated the relationship between the function of ABCB4 and lipid rafts using mouse canalicular membranes and HEK293 cells stably expressing ABCB4. ABCB4 and ABCB1 were mainly distributed in nonraft membranes. The expression of ABCB4, but not ABCB1, led to significant increases in the phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) contents in nonraft membranes and further enrichment of SM and cholesterol in raft membranes. The ABCB4-mediated efflux of PC, PE, and SM was significantly stimulated by taurocholate, while the efflux of PE and SM was much less than that of PC. This ABCB4-mediated efflux was completely abolished by BODIPY-verapamil, which hardly partitioned into raft membranes. In addition, ABCB1 and ABCB4 mediated the efflux of rhodamine 123 and rhodamine 6G from nonraft membranes, which was not affected by taurocholate. We conclude that ABCB4 located in nonrafts, but not in rafts, is predominantly involved in the efflux of phospholipids and other substrates.

  12. Bile salt-stimulated phospholipid efflux mediated by ABCB4 localized in nonraft membranes

    PubMed Central

    Morita, Shin-ya; Tsuda, Tadanori; Horikami, Manami; Teraoka, Reiko; Kitagawa, Shuji; Terada, Tomohiro

    2013-01-01

    ABCB4 is necessary for the secretion of phospholipids from hepatocytes into bile and for the protection of cell membranes against bile salts. Lipid rafts are plasma membrane microdomains containing high contents of cholesterol and sphingolipids, which are separated by Triton X-100 extraction or OptiPrep gradient centrifugation. In this study, we investigated the relationship between the function of ABCB4 and lipid rafts using mouse canalicular membranes and HEK293 cells stably expressing ABCB4. ABCB4 and ABCB1 were mainly distributed in nonraft membranes. The expression of ABCB4, but not ABCB1, led to significant increases in the phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) contents in nonraft membranes and further enrichment of SM and cholesterol in raft membranes. The ABCB4-mediated efflux of PC, PE, and SM was significantly stimulated by taurocholate, while the efflux of PE and SM was much less than that of PC. This ABCB4-mediated efflux was completely abolished by BODIPY-verapamil, which hardly partitioned into raft membranes. In addition, ABCB1 and ABCB4 mediated the efflux of rhodamine 123 and rhodamine 6G from nonraft membranes, which was not affected by taurocholate. We conclude that ABCB4 located in nonrafts, but not in rafts, is predominantly involved in the efflux of phospholipids and other substrates. PMID:23468132

  13. Sub-mitochondrial localization of the genetic-tagged mitochondrial intermembrane space-bridging components Mic19, Mic60 and Sam50.

    PubMed

    Sastri, Mira; Darshi, Manjula; Mackey, Mason; Ramachandra, Ranjan; Ju, Saeyeon; Phan, Sebastien; Adams, Stephen; Stein, Kathryn; Douglas, Christopher R; Kim, Jiwan John; Ellisman, Mark H; Taylor, Susan S; Perkins, Guy A

    2017-10-01

    Each mitochondrial compartment contains varying protein compositions that underlie a diversity of localized functions. Insights into the localization of mitochondrial intermembrane space-bridging (MIB) components will have an impact on our understanding of mitochondrial architecture, dynamics and function. By using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and performing electron tomography, we have mapped at nanoscale resolution three key MIB components, Mic19, Mic60 and Sam50 (also known as CHCHD3, IMMT and SAMM50, respectively), in the environment of structural landmarks such as cristae and crista junctions (CJs). Tagged Mic19 and Mic60 were located at CJs, distributed in a network pattern along the mitochondrial periphery and also enriched inside cristae. We discovered an association of Mic19 with cytochrome c oxidase subunit IV. It was also found that tagged Sam50 is not uniformly distributed in the outer mitochondrial membrane and appears to incompletely overlap with Mic19- or Mic60-positive domains, most notably at the CJs. © 2017. Published by The Company of Biologists Ltd.

  14. Cytochrome b 6 f function and localization, phosphorylation state of thylakoid membrane proteins and consequences on cyclic electron flow.

    PubMed

    Dumas, Louis; Chazaux, Marie; Peltier, Gilles; Johnson, Xenie; Alric, Jean

    2016-09-01

    Both the structure and the protein composition of thylakoid membranes have an impact on light harvesting and electron transfer in the photosynthetic chain. Thylakoid membranes form stacks and lamellae where photosystem II and photosystem I localize, respectively. Light-harvesting complexes II can be associated to either PSII or PSI depending on the redox state of the plastoquinone pool, and their distribution is governed by state transitions. Upon state transitions, the thylakoid ultrastructure and lateral distribution of proteins along the membrane are subject to significant rearrangements. In addition, quinone diffusion is limited to membrane microdomains and the cytochrome b 6 f complex localizes either to PSII-containing grana stacks or PSI-containing stroma lamellae. Here, we discuss possible similarities or differences between green algae and C3 plants on the functional consequences of such heterogeneities in the photosynthetic electron transport chain and propose a model in which quinones, accepting electrons either from PSII (linear flow) or NDH/PGR pathways (cyclic flow), represent a crucial control point. Our aim is to give an integrated description of these processes and discuss their potential roles in the balance between linear and cyclic electron flows.

  15. C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

    NASA Astrophysics Data System (ADS)

    Russ, K. A.; Elvati, P.; Parsonage, T. L.; Dews, A.; Jarvis, J. A.; Ray, M.; Schneider, B.; Smith, P. J. S.; Williamson, P. T. F.; Violi, A.; Philbert, M. A.

    2016-02-01

    There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern

  16. Both idebenone and idebenol are localized near the lipid-water interface of the membrane and increase its fluidity.

    PubMed

    Gómez-Murcia, Victoria; Torrecillas, Alejandro; de Godos, Ana M; Corbalán-García, Senena; Gómez-Fernández, Juan C

    2016-06-01

    Idebenone is a synthetic analog of coenzyme Q; both share a quinone moiety but idebenone has a shorter lipophilic tail ending with a hydroxyl group. Differential scanning calorimetry experiments showed that both idebenone and idebenol widened and shifted the phase transition of 1,2-dipalmitoylphosphatidylcholine (DPPC) to a lower temperature and a phase separation with different concentrations of these molecules was observed. Also small angle X-ray diffraction and wide angle X-ray diffraction revealed that both, idebenone and idebenol, induced laterally separated phases in fluid membranes when included in DPPC membranes. Electronic profiles showed that both forms, idebenone and idebenol, reduced the thickness of the fluid membrane. (2)H NMR measurements showed that the order of the membrane decreased at all temperatures in the presence of idebenone or idebenol, the greatest disorder being observed in the segments of the acyl chains close to the lipid-water interface. (1)H NOESY MAS NMR spectra were obtained using 1-palmitoyl-2-oleoyl-phosphatidylcholine membranes and results pointed to a similar location in the membrane for both forms, with the benzoquinone or benzoquinol rings and their terminal hydroxyl group of the hydrophobic chain located near the lipid/water interface of the phospholipid bilayer and the terminal hydroxyl group of the hydrophobic chain of both compounds located at the lipid/water interface. Taken together, all these different locations might explain the different physiological behavior shown by the idebenone/idebenol compared with the ubiquinone-10/ubiquinol-10 pair in which both compounds are differently localized in the membrane. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages.

    PubMed

    Russ, K A; Elvati, P; Parsonage, T L; Dews, A; Jarvis, J A; Ray, M; Schneider, B; Smith, P J S; Williamson, P T F; Violi, A; Philbert, M A

    2016-02-21

    There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.

  18. C60 Fullerene Localization and Membrane Interactions in RAW 264.7 Immortalized Mouse Macrophages

    PubMed Central

    Russ, K. A.; Elvati, P.; Parsonage, T. L.; Dews, A.; Jarvis, J. A.; Ray, M.; Schneider, B.; Smith, P. J. S.; Williamson, P. T. F.; Violi, A.; Philbert, M. A.

    2016-01-01

    There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis. PMID:26866469

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

    PubMed Central

    Craven, Ryan

    2015-01-01

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

  20. Structural change of bacteriorhodopsin in the purple membrane above pH 10 decreases heterogeneity of the irreversible photobleaching components.

    PubMed

    Yokoyama, Yasunori; Sonoyama, Masashi; Nakano, Tatsuhiko; Mitaku, Shigeki

    2007-09-01

    Kinetic investigations of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at high temperature have previously shown two kinds of bR species upon light illumination. The bR species consist of kinetically fast- and slow-denatured components, whose proportions were dependent upon structural changes in dark, as shown by CD. In order to elucidate electrostatic contribution on the heterogeneous stability and the bR structure in PM, photobleaching behaviour and structural changes over a wide pH range were investigated by kinetics as well as various spectroscopic techniques. Kinetics revealed that photobleaching below pH 9 obeyed double-exponential functions, whereas measurements above pH 10 were characterized by a single-decay component. FT-IR deconvoluted spectra showed a alpha(II)-to-alpha(I) transition in the transmembrane helices around pH 10. Near-IR Raman scattering spectra demonstrated the equilibrium shift of retinal isomers from all trans to 13-cis form. Near-UV CD spectra suggested configurational changes in the aromatic residues around the retinal pocket. An exciton-to-positive transition in visible CD spectrum was observed. This indicates disorganization in the 2D-crystalline lattice of PM, which occurred concomitantly with the changes above pH 10. A model for the changes in kinetic behaviour and molecular structure around pH 10 is discussed, focusing on changes in charge distribution upon alkalinization.

  1. Relative roles of local and reflex components in cutaneous vasoconstriction during skin cooling in humans.

    PubMed

    Alvarez, Guy E; Zhao, Kun; Kosiba, Wojciech A; Johnson, John M

    2006-06-01

    The reduction in skin blood flow (SkBF) with cold exposure is partly due to the reflex vasoconstrictor response from whole body cooling (WBC) and partly to the direct effects of local cooling (LC). Although these have been examined independently, little is known regarding their roles when acting together, as occurs in environmental cooling. We tested the hypothesis that the vasoconstrictor response to combined LC and WBC would be additive, i.e., would equal the sum of their independent effects. We further hypothesized that LC would attenuate the reflex vasoconstrictor response to WBC. We studied 16 (7 women, 9 men) young (30.5+/-2 yr) healthy volunteers. LC and WBC were accomplished with metal Peltier cooler-heater probe holders and water-perfused suits, respectively. Forearm SkBF was monitored by laser-Doppler flowmetry (LDF). Cutaneous vascular conductance (CVC) was calculated as LDF/blood pressure. Subjects underwent 15 min of LC alone or 15 min of WBC with and without simultaneous LC, either at equal levels (34-31 degrees C) or as equipotent stimuli (34-28 degrees C LC; 34-31 degrees C WBC). The fall in CVC with combined WBC and LC was greater (P<0.05) than for either alone (57.0+/-5% combined vs. 39.2+/-6% WBC; 34.4+/-4% LC) with equipotent cooling, but it was only significantly greater than for LC alone with equal levels of cooling (51.3+/-8% combined vs. 29.5+/-4% LC). The sum of the independent effects of WBC and LC was greater than their combined effects (74.9+/-4 vs. 51.3+/-8% equal and 73.6+/-7 vs. 57.0+/-5% equipotent; P<0.05). The fall in CVC with WBC at LC sites was reduced compared with control sites (17.6+/-2 vs. 42.4+/-8%; P<0.05). Hence, LC contributes importantly to the reduction in SkBF with body cooling, but also suppresses the reflex response, resulting in a nonadditive effect of these two components.

  2. The Relationship between Components of the Ohio Local School District Report Card and the Outcome of a School Tax Levy

    ERIC Educational Resources Information Center

    Wheatley, Vicki Ann

    2012-01-01

    The relationship between components of the local school district report card, school district typology, and the outcome of public school tax levy requests were examined in this study. A correlation research design was used to measure the relationship between the independent variables (performance index, average yearly progress, value added,…

  3. The Relationship between Components of the Ohio Local School District Report Card and the Outcome of a School Tax Levy

    ERIC Educational Resources Information Center

    Wheatley, Vicki Ann

    2012-01-01

    The relationship between components of the local school district report card, school district typology, and the outcome of public school tax levy requests were examined in this study. A correlation research design was used to measure the relationship between the independent variables (performance index, average yearly progress, value added,…

  4. Historical review: the carbon monoxide diffusing capacity (DLCO) and its membrane (DM) and red cell (Theta.Vc) components.

    PubMed

    Hughes, J M B; Bates, D V

    2003-11-14

    The single breath carbon monoxide diffusing capacity (DLCO sb), also called the transfer factor (TLCO), was introduced by Marie and August Krogh in two papers (Krogh and Krogh, Skand. Arch. Physiol. 23, 236-247, 1909; Krogh, J. Physiol., Lond. 49, 271-296, 1915). Physiologically, their measurements showed that sufficient oxygen (by extrapolation from CO) diffused passively from gas to blood without the need to postulate oxygen secretion, a popular theory at the time. Their DLCO sb technique was neglected until the advent of the infra-red CO meter in the 1950s. Ogilvie et al., J. Clin. Invest. 36, 1-17, 1957 published a standardized technique for a 'modified Krogh' single breath DLCO, which eventually became the method of choice in pulmonary function laboratories. The Roughton-Forster equation (J. Appl. Physiol. 1957, 11, 290-302) was an important step conceptually; it partitioned alveolar-capillary diffusion of oxygen (O2) and carbon monoxide (CO) into a membrane component (DM) and a red cell component (theta.Vc) where theta is the DLCO (or DL(O2)) per ml of blood (measured in vitro), and Vc is the pulmonary capillary volume. This equation was based on the kinetics of O2 and CO with haemoglobin (Hb) in solution and with whole blood Hartridge and Roughton, Nature, 1923, 111, 325-326; Proc. R. Soc. Lond. Ser. A, 1923, 104, 376-394; (Proc. R. Soc. Lond. Ser. B, 1923, 94, 336-367; Proc. R. Soc. Lond. Ser. A 1923, 104, 395-430; J. Physiol., Lond. 1927, 62, 232-242; Roughton, Proc. R. Soc. Lond. Ser. B 1932, 111, 1-36) and on the relationship between alveolar P(O2) and 1/DLCO. Subsequently, the relationship between DL(O2) (Lilienthal et al., Am. J. Physiol. 147, 199-216, 1946) and DL(CO) was defined. More recently, the measurement of the nitric oxide diffusing capacity (DLNO) has been introduced. For DL(O2) and DLNO the membrane component (as 1/DM) is an important part of the overall diffusion (transfer) resistance. For the DLCO, 1/theta.Vc probably plays the greater

  5. Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: theory and application to membrane proteins.

    PubMed

    Gopinath, T; Mote, Kaustubh R; Veglia, Gianluigi

    2011-08-21

    NMR anisotropic parameters such as dipolar couplings and chemical shifts are central to structure and orientation determination of aligned membrane proteins and liquid crystals. Among the separated local field experiments, the proton evolved local field (PELF) scheme is particularly suitable to measure dynamically averaged dipolar couplings and give information on local molecular motions. However, the PELF experiment requires the acquisition of several 2D datasets at different mixing times to optimize the sensitivity for the complete range of dipolar couplings of the resonances in the spectrum. Here, we propose a new PELF experiment that takes the advantage of the Hadamard encoding (HE) to obtain higher sensitivity for a broad range of dipolar couplings using a single 2D experiment. The HE scheme is obtained by selecting the spin operators with phase switching of hard pulses. This approach enables one to detect four spin operators, simultaneously, which can be processed into two 2D spectra covering a broader range of dipolar couplings. The advantages of the new approach are illustrated for a U-(15)N NAL single crystal and the U-(15)N labeled single-pass membrane protein sarcolipin reconstituted in oriented lipid bicelles. The HE-PELF scheme can be implemented in other multidimensional experiments to speed up the characterization of the structure and dynamics of oriented membrane proteins and liquid crystalline samples.

  6. Analysis of the localization and topology of nurim, a polytopic protein tightly associated with the inner nuclear membrane.

    PubMed

    Hofemeister, Helmut; O'Hare, Peter

    2005-01-28

    Nurim is an inner nuclear membrane (INM) protein that was first isolated in a visual screen for nuclear envelope-localizing proteins. Nurim lacks an N-terminal domain characteristic of other INM proteins examined to date and may represent a class of proteins that localize to the INM by a distinct mechanism. To further characterize this protein, we constructed nurim-green fluorescent protein fusions and analyzed aspects of localization, biochemistry, and membrane topology. Results from immunoprobing and protease protection assays together with other analyses indicate that nurim (total length of 262 residues) is a six transmembrane-spanning protein and contains a hairpin turn in its C-terminal transmembrane domain, resulting in the N and C termini residing on the same side of the membrane. A loop region between the fourth and fifth transmembrane domains is exposed toward the nucleoplasm and contains a region accessible for site-specific endoproteinase cleavage. In biochemical fractionation, nurim remained extremely tightly bound to nuclear fractions and was released in significant quantities only in the presence of 4 m urea. Under conditions in which nuclear lamins were completely extracted, a significant population of nurim remained resistant to solubilization. This tight binding requires the C-terminal region of the protein. DNase treatment only marginally influenced its retention characteristics in nuclei. Results from consideration of sequence alignments and identification of specific topological features of nurim indicate that it may possess enzymic function. These results are discussed with reference to the retention mechanism and possible nuclear function of nurim.

  7. Determination of the depth of localized radioactive contamination by 137Cs and 60Co in sand with principal component analysis.

    PubMed

    Adams, Jamie C; Mellor, Matthew; Joyce, Malcolm J

    2011-10-01

    A method to determine the depth of buried localized radioactive contamination nonintrusively and nondestructively using principal component analysis is described. The γ-ray spectra from two radionuclides, cesium-137 and cobalt-60, have been analyzed to derive the two principal components that change most significantly as a result of varying the depth of the sources in a bespoke sand-filled phantom. The relationship between depth (d) and the angle (θ) between the first two principal component coefficients has been derived for both cases, viz. d(Φ) = x + y log(e) Φ where x and y are constants dependent on the shielding material and the γ-ray energy spectrum of the radioactivity in question, and φ is a function of θ. The technique enables the depth of a localized radioactive source to be determined nonintrusively in the range 5 to 50 mm with an accuracy of ±1 mm.

  8. Goodpasture antigen of the glomerular basement membrane: localization to noncollagenous regions of type IV collagen.

    PubMed Central

    Wieslander, J; Barr, J F; Butkowski, R J; Edwards, S J; Bygren, P; Heinegård, D; Hudson, B G

    1984-01-01

    The glomerular basement membrane antigen in Goodpasture syndrome is a collagenase-resistant molecule with a monomer molecular weight of about 26,000. Type IV collagen isolated from glomerular basement membrane contains collagenase-resistant sequences within its structure. Polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and chemical analysis were used to demonstrate that the collagenase-resistant sequences of type IV collagen contain Goodpasture antigen. Images PMID:6328527

  9. A method for detergent-free isolation of membrane proteins in their local lipid environment.

    PubMed

    Lee, Sarah C; Knowles, Tim J; Postis, Vincent L G; Jamshad, Mohammed; Parslow, Rosemary A; Lin, Yu-Pin; Goldman, Adrian; Sridhar, Pooja; Overduin, Michael; Muench, Stephen P; Dafforn, Timothy R

    2016-07-01

    Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

  10. The β-Lactam Resistance Protein Blr, a Small Membrane Polypeptide, Is a Component of the Escherichia coli Cell Division Machinery

    PubMed Central

    Karimova, Gouzel; Davi, Marilyne

    2012-01-01

    In Escherichia coli, cell division is performed by a multimolecular machinery called the divisome, made of 10 essential proteins and more than 20 accessory proteins. Through a bacterial two-hybrid library screen, we identified the E. coli β-lactam resistance protein Blr, a short membrane polypeptide of 41 residues, as an interacting partner of the essential cell division protein FtsL. In addition to FtsL, Blr was found to associate with several other divisomal proteins, including FtsI, FtsK, FtsN, FtsQ, FtsW, and YmgF. Using fluorescently tagged Blr, we showed that this peptide localizes to the division septum and that its colocalization requires the presence of the late division protein FtsN. Although Blr is not essential, previous studies have shown that the inactivation of the blr gene increased the sensitivity of bacteria to β-lactam antibiotics or their resistance to cell envelope stress. Here, we found that Blr, when overproduced, restores the viability of E. coli ftsQ1(Ts) cells, carrying a thermosensitive allele of the ftsQ gene, during growth under low-osmotic-strength conditions (e.g., in synthetic media or in Luria-Bertani broth without NaCl). In contrast, the inactivation of blr increases the osmosensitivity of ftsQ1(Ts) cells, and blr ftsQ1 double mutants exhibit filamentous growth in LB broth even at a moderate salt concentration (0.5% NaCl) compared to parental ftsQ1(Ts) cells. Altogether, our results suggest that the small membrane polypeptide Blr is a novel component of the E. coli cell division apparatus involved in the stabilization of the divisome under certain stress conditions. PMID:22885295

  11. Radiographic contrast media alterate the localization of actin/band4.9 in the membrane cytoskeleton of human erythrocytes.

    PubMed

    Franke, R P; Scharnweber, T; Fuhrmann, R; Mrowietz, C; Wenzel, F; Krüger, A; Jung, F

    2014-01-01

    Different radiographic contrast media (RCM) were shown to induce morphological changes of blood cells (e.g. erythrocytes or thrombocytes) and endothelial cells. The echinocytic shape change of erythrocytes, particularly, affords alterations of the membrane cytoskeleton. The cytoskeleton plays a crucial role for the shape and deformability of the red blood cell. Disruption of the interaction between components of the red blood cell membrane cytoskeleton may cause a loss of structural and functional integrity of the membrane. In this study band4.9 and actin as components of the cytoskeletal junctional complex were examined in human erythrocytes after suspension in autologous plasma or in plasma RCM mixtures (30% v/v Iodixanol-320 or Iopromide-370) followed by a successive double staining with TRITC-/FITC-coupled monoclonal antibodies. After adding Iopromide-370 to the plasma in practically none of the cells the rounded conformation of the membrane cytoskeleton - as it appeared in cells suspended in autologous plasma - was found. In addition, Iopromide-370 induced thin lines and coarse knob-like structures of band4.9 at the cell periphery while most cell centers were devoid of band4.9, and a box-like arrangement of bands of band4.9. A dissociation between colours red (actin) and green (band4.9) occurred as well. In contrast, erythrocytes suspended in a plasma/Iodixanol-320 mixture showed a membrane cytoskeleton comparable to cells suspended in autologous plasma, Similar results were found with respect to the distribution of actin. This study revealed for the first time RCM-dependent differences in band4.9 activities as possible pathophysiological mechanism for the chemotoxicity of radiographic contrast media.

  12. The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth

    PubMed Central

    Dickinson, Amanda J. G.; Sive, Hazel L.

    2009-01-01

    Summary The primary mouth forms from ectoderm and endoderm at the extreme anterior of the embryo, a conserved mesoderm-free region. In Xenopus, a very early step in primary mouth formation is loss of the basement membrane between the ectoderm and endoderm. In an unbiased microarray screen, we defined genes encoding the sFRPs Frzb-1 and Crescent as transiently and locally expressed in the primary mouth anlage. Using antisense oligonucleotides and `face transplants', we show that frzb-1 and crescent expression is specifically required in the primary mouth region at the time this organ begins to form. Several assays indicate that Frzb-1 and Crescent modulate primary mouth formation by suppressing Wnt signaling, which is likely to be mediated by β-catenin. First, a similar phenotype (no primary mouth) is seen after loss of Frzb-1/Crescent function to that seen after temporally and spatially restricted overexpression of Wnt-8. Second, overexpression of either Frzb-1 or Dkk-1 results in an enlarged primary mouth anlage. Third, overexpression of Dkk-1 can restore a primary mouth to embryos in which Frzb-1/Crescent expression has been inhibited. We show that Frzb-1/Crescent function locally promotes basement membrane dissolution in the primary mouth primordium. Consistently, Frzb-1 overexpression decreases RNA levels of the essential basement membrane genes fibronectin and laminin, whereas Wnt-8 overexpression increases the levels of these RNAs. These data are the first to connect Wnt signaling and basement membrane integrity during primary mouth development, and suggest a general paradigm for the regulation of basement membrane remodeling. PMID:19224982

  13. Nanoscale Effects of Ethanol and Naltrexone on Protein Organization in the Plasma Membrane Studied by Photoactivated Localization Microscopy (PALM)

    PubMed Central

    Tobin, Steven J.; Cacao, Eliedonna E.; Hong, Daniel Wing Wo; Terenius, Lars; Vukojevic, Vladana; Jovanovic-Talisman, Tijana

    2014-01-01

    Background Ethanol affects the signaling of several important neurotransmitter and neuromodulator systems in the CNS. It has been recently proposed that ethanol alters the dynamic lateral organization of proteins and lipids in the plasma membrane, thereby affecting surface receptor-mediated cellular signaling. Our aims are to establish whether pharmacologically relevant levels of ethanol can affect the lateral organization of plasma membrane and cytoskeletal proteins at the nanoscopic level, and investigate the relevance of such perturbations for mu-opioid receptor (MOP) function. Methodology/Principal Findings We used Photoactivated Localization Microscopy with pair-correlation analysis (pcPALM), a quantitative fluorescence imaging technique with high spatial resolution (15–25 nm) and single-molecule sensitivity, to study ethanol effects on protein organization in the plasma membrane. We observed that short (20 min) exposure to 20 and 40 mM ethanol alters protein organization in the plasma membrane of cells that harbor endogenous MOPs, causing a rearrangement of the lipid raft marker glycosylphosphatidylinositol (GPI). These effects could be largely occluded by pretreating the cells with the MOP antagonist naltrexone (200 nM for 3 hours). In addition, ethanol induced pronounced actin polymerization, leading to its partial co-localization with GPI. Conclusions/Significance Pharmacologically relevant levels of ethanol alter the lateral organization of GPI-linked proteins and induce actin cytoskeleton reorganization. Pretreatment with the MOP antagonist naltrexone is protective against ethanol action and significantly reduces the extent to which ethanol remodels the lateral organization of lipid-rafts-associated proteins in the plasma membrane. Super-resolution pcPALM reveals details of ethanol action at the nanoscale level, giving new mechanistic insight on the cellular and molecular mechanisms of its action. PMID:24503624

  14. The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth.

    PubMed

    Dickinson, Amanda J G; Sive, Hazel L

    2009-04-01

    The primary mouth forms from ectoderm and endoderm at the extreme anterior of the embryo, a conserved mesoderm-free region. In Xenopus, a very early step in primary mouth formation is loss of the basement membrane between the ectoderm and endoderm. In an unbiased microarray screen, we defined genes encoding the sFRPs Frzb-1 and Crescent as transiently and locally expressed in the primary mouth anlage. Using antisense oligonucleotides and ;face transplants', we show that frzb-1 and crescent expression is specifically required in the primary mouth region at the time this organ begins to form. Several assays indicate that Frzb-1 and Crescent modulate primary mouth formation by suppressing Wnt signaling, which is likely to be mediated by beta-catenin. First, a similar phenotype (no primary mouth) is seen after loss of Frzb-1/Crescent function to that seen after temporally and spatially restricted overexpression of Wnt-8. Second, overexpression of either Frzb-1 or Dkk-1 results in an enlarged primary mouth anlage. Third, overexpression of Dkk-1 can restore a primary mouth to embryos in which Frzb-1/Crescent expression has been inhibited. We show that Frzb-1/Crescent function locally promotes basement membrane dissolution in the primary mouth primordium. Consistently, Frzb-1 overexpression decreases RNA levels of the essential basement membrane genes fibronectin and laminin, whereas Wnt-8 overexpression increases the levels of these RNAs. These data are the first to connect Wnt signaling and basement membrane integrity during primary mouth development, and suggest a general paradigm for the regulation of basement membrane remodeling.

  15. Phospholipid flippase activities and substrate specificities of human type IV P-type ATPases localized to the plasma membrane.

    PubMed

    Takatsu, Hiroyuki; Tanaka, Gaku; Segawa, Katsumori; Suzuki, Jun; Nagata, Shigekazu; Nakayama, Kazuhisa; Shin, Hye-Won

    2014-11-28

    Type IV P-type ATPases (P4-ATPases) are believed to translocate aminophospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. The yeast P4-ATPases, Drs2p and Dnf1p/Dnf2p, flip nitrobenzoxadiazole-labeled phosphatidylserine at the Golgi complex and nitrobenzoxadiazole-labeled phosphatidylcholine (PC) at the plasma membrane, respectively. However, the flippase activities and substrate specificities of mammalian P4-ATPases remain incompletely characterized. In this study, we established an assay for phospholipid flippase activities of plasma membrane-localized P4-ATPases using human cell lines stably expressing ATP8B1, ATP8B2, ATP11A, and ATP11C. We found that ATP11A and ATP11C have flippase activities toward phosphatidylserine and phosphatidylethanolamine but not PC or sphingomyelin. By contrast, ATPase-deficient mutants of ATP11A and ATP11C did not exhibit any flippase activity, indicating that these enzymes catalyze flipping in an ATPase-dependent manner. Furthermore, ATP8B1 and ATP8B2 exhibited preferential flippase activities toward PC. Some ATP8B1 mutants found in patients of progressive familial intrahepatic cholestasis type 1 (PFIC1), a severe liver disease caused by impaired bile flow, failed to translocate PC despite their delivery to the plasma membrane. Moreover, incorporation of PC mediated by ATP8B1 can be reversed by simultaneous expression of ABCB4, a PC floppase mutated in PFIC3 patients. Our findings elucidate the flippase activities and substrate specificities of plasma membrane-localized human P4-ATPases and suggest that phenotypes of some PFIC1 patients result from impairment of the PC flippase activity of ATP8B1. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Mechanism of specific membrane targeting by C2 domains: localized pools of target lipids enhance Ca2+ affinity.

    PubMed

    Corbin, John A; Evans, John H; Landgraf, Kyle E; Falke, Joseph J

    2007-04-10

    membrane, which provides a high local concentration of target lipid, is the effective Ca2+ affinity increased by the coupled binding equilibrium to a level that enables substantial Ca2+ activation and target docking. Overall, the findings emphasize the importance of using physiological ligand concentrations in targeting studies because super-physiological concentrations can drive docking interactions even when an important targeting molecule is missing.

  17. Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

    PubMed

    Seipold, Lisa; Damme, Markus; Prox, Johannes; Rabe, Björn; Kasparek, Petr; Sedlacek, Radislav; Altmeppen, Hermann; Willem, Michael; Boland, Barry; Glatzel, Markus; Saftig, Paul

    2017-01-01

    Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.

  18. Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes

    PubMed Central

    1993-01-01

    , the infiltrating T cells could be stained with mAbs to alpha 3 or beta 1 and were localized in close proximity to the epiligrin-containing basement membrane. Infiltrating lymphocytes in malignant cutaneous B disease (CBCL) did not express alpha 3 beta 1 by immunohistochemical techniques and did not associate with the epidermal basement membrane. The present findings clearly define a function for alpha 3 beta 1 in T cells and strongly suggest that alpha 3 beta 1 interaction with epiligrin may be involved in the pathogenesis of cutaneous inflammation. PMID:8501119

  19. Membrane-localized β-subunits alter the PIP2 regulation of high-voltage activated Ca2+ channels.

    PubMed

    Suh, Byung-Chang; Kim, Dong-Il; Falkenburger, Björn H; Hille, Bertil

    2012-02-21

    The β-subunits of voltage-gated Ca(2+) (Ca(V)) channels regulate the functional expression and several biophysical properties of high-voltage-activated Ca(V) channels. We find that Ca(V) β-subunits also determine channel regulation by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)). When Ca(V)1.3, -2.1, or -2.2 channels are cotransfected with the β3-subunit, a cytosolic protein, they can be inhibited by activating a voltage-sensitive lipid phosphatase to deplete PIP(2). When these channels are coexpressed with a β2a-subunit, a palmitoylated peripheral membrane protein, the inhibition is much smaller. PIP(2) sensitivity could be increased by disabling the two palmitoylation sites in the β2a-subunit. To further test effects of membrane targeting of Ca(V) β-subunits on PIP(2) regulation, the N terminus of Lyn was ligated onto the cytosolic β3-subunit to confer lipidation. This chimera, like the Ca(V) β2a-subunit, displayed plasma membrane localization, slowed the inactivation of Ca(V)2.2 channels, and increased the current density. In addition, the Lyn-β3 subunit significantly decreased Ca(V) channel inhibition by PIP(2) depletion. Evidently lipidation and membrane anchoring of Ca(V) β-subunits compete with the PIP(2) regulation of high-voltage-activated Ca(V) channels. Compared with expression with Ca(V) β3-subunits alone, inhibition of Ca(V)2.2 channels by PIP(2) depletion could be significantly attenuated when β2a was coexpressed with β3. Our data suggest that the Ca(V) currents in neurons would be regulated by membrane PIP(2) to a degree that depends on their endogenous β-subunit combinations.

  20. Quantitative image analysis tool to study the plasma membrane localization of proteins and cortical actin in neuroendocrine cells.

    PubMed

    Kurps, Julia; Broeke, Jurjen H; Cijsouw, Tony; Kompatscher, Andreas; van Weering, Jan R T; de Wit, Heidi

    2014-10-30

    Adrenal chromaffin cells are a widely used model system to study regulated exocytosis and other membrane-associated processes. Alterations in the amount and localization of the proteins involved in these processes can be visualized with fluorescent probes that report the effect of different stimuli or genetic modifications. However, the quantitative analysis of such images remains difficult, especially when focused on specific locations, such as the plasma membrane. We developed an image analysis algorithm, named plasma membrane analysis in chromaffin cells (PlasMACC). PlasMACC enables automatic detection of the plasma membrane region and quantitative analysis of multi-fluorescent signals from spherical cells. PlasMACC runs in the image analysis software ImageJ environment, it is user-friendly and freely available. PlasMACC delivers detailed information about intensity, thickness and density of fluorescent signals at the plasma membrane of both living and fixed cells. Individual signals can be compared between cells and different signals within one cell can be correlated. PlasMACC can process conventional laser-scanning confocal images as well as data obtained by super-resolution methods such as structured illumination microscopy. By comparing PlasMACC to methods currently used in the field, we show more consistent quantitative data due to the fully automated algorithm. PlasMACC also provides an expanded set of novel analysis parameters. PlasMACC enables a detailed quantification of fluorescent signals at the plasma membrane of spherical cells in an unbiased and reliable fashion. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Screening anti-allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC-ESI-MS/MS method.

    PubMed

    Lv, Yanni; Sun, Yueming; Fu, Jia; Kong, Liyun; Han, Shengli

    2017-02-01

    Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti-allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC-ESI-MS/MS method was developed to screen, analyze and identify the anti-allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC-ESI-MS/MS. The coupled system was then used to screen anti-allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose-dependent manner from 1 to 100 μm. The LAD2 cell membrane chromatography online with UHPLC-ESI-MS/MS method is an effective technique for screening the anti-allergic components of Huangqi.

  2. A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus.

    PubMed

    Resch, Ulrike; Tsatsaronis, James Anthony; Le Rhun, Anaïs; Stübiger, Gerald; Rohde, Manfred; Kasvandik, Sergo; Holzmeister, Susanne; Tinnefeld, Philip; Wai, Sun Nyunt; Charpentier, Emmanuelle

    2016-11-01

    Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to "anchorless surface proteins." Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence

  3. 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

  4. Localization of a Mg2+-activated ATPase in the plasma membrane of Trypanosoma cruzi.

    PubMed

    Meirelles, M N; De Souza, W

    1984-02-01

    The Wachstein and Meisel incubation medium was used to detect ATPase activity in epimastigote, spheromastigote (amastigote), and bloodstream trypomastigote forms of Trypanosoma cruzi. Reaction product, indicative of enzyme activity, was associated with the plasma membrane covering the cell body and the flagellum of the parasite. No reaction product was found in the portion of the plasma membrane lining the flagellar pocket. The plasma membrane-associated ATPase activity was not inhibited by ouabain or oligomycin, was detected in incubation medium without K+, was inhibited by prolonged glutaraldehyde fixation, and its activity was diminished when Mg2+ was omitted from the incubation medium. The Ernst medium was used to detect Na+-K+-ATPase activity in T. cruzi. No reaction product indicative of the presence of this enzyme was detected. Reaction product indicative of 5'-nucleotidase was not detected in T. cruzi. Acid phosphatase activity was detected in lysosomes. Those results indicate that a Mg2+-activated ATPase is present in the plasma membrane of T. cruzi and that it can be used as an enzyme marker, provided that the mitochondrial and flagellar ATPases are inhibited, to assess the purity of plasma membrane fractions isolated from this parasite.

  5. Sustained Attention to