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Sample records for photosynthetic membrane expression

  1. Oxygen dynamics in photosynthetic membranes.

    NASA Astrophysics Data System (ADS)

    Savikhin, Sergei; Kihara, Shigeharu

    2008-03-01

    Production of oxygen by oxygenic photosynthetic organisms is expected to raise oxygen concentration within their photosynthetic membranes above normal aerobic values. These raised levels of oxygen may affect function of many proteins within photosynthetic cells. However, experiments on proteins in vitro are usually performed in aerobic (or anaerobic) conditions since the oxygen content of a membrane is not known. Using theory of diffusion and measured oxygen production rates we estimated the excess levels of oxygen in functioning photosynthetic cells. We show that for an individual photosynthetic cell suspended in water oxygen level is essentially the same as that for a non-photosynthetic sell. These data suggest that oxygen protection mechanisms may have evolved after the development of oxygenic photosynthesis in primitive bacteria and was driven by the overall rise of oxygen concentration in the atmosphere. Substantially higher levels of oxygen are estimated to occur in closely packed colonies of photosynthetic bacteria and in green leafs.

  2. Electrostatics of photosynthetic reaction centers in membranes.

    PubMed

    Pennisi, Cristian P; Greenbaum, Elias; Yoshida, Ken

    2006-01-01

    Photosynthetic reaction centers are integral membrane complexes. They have potential application as molecular photovoltaic structures and have been used in diverse technological applications. A three-dimensional electrostatic model of the photosystem I reaction center (PSI) embedded in a lipid membrane is presented. The potential is obtained by solving the Poisson-Boltzmann equation with the finite element method (FEM). Simulations showing the potential distribution in a vesicle containing PSI reaction centers under different conditions are presented. The results of the simulations are compared with previous findings and a possible application of PSI to provide light activation of voltage-gated ion channels is discussed. PMID:17946611

  3. A Model for Prediction of Heat Stability of Photosynthetic Membranes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A previous study has revealed a positive correlation between heat-induced damage to photosynthetic membranes (thylakoid membranes) and chlorophyll loss. In this study, we exploited this correlation and developed a model for prediction of thermal damage to thylakoids. Prediction is based on estimat...

  4. Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes*

    PubMed Central

    Tietz, Stefanie; Puthiyaveetil, Sujith; Enlow, Heather M.; Yarbrough, Robert; Wood, Magnus; Semchonok, Dmitry A.; Lowry, Troy; Li, Zhirong; Jahns, Peter; Boekema, Egbert J.; Lenhert, Steven; Niyogi, Krishna K.; Kirchhoff, Helmut

    2015-01-01

    The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion. PMID:25897076

  5. Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes.

    PubMed

    Tietz, Stefanie; Puthiyaveetil, Sujith; Enlow, Heather M; Yarbrough, Robert; Wood, Magnus; Semchonok, Dmitry A; Lowry, Troy; Li, Zhirong; Jahns, Peter; Boekema, Egbert J; Lenhert, Steven; Niyogi, Krishna K; Kirchhoff, Helmut

    2015-05-29

    The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion. PMID:25897076

  6. A method for estimation of permittivity in photosynthetic membranes and the effect of permittivity on the photosynthetic quantum yield

    NASA Astrophysics Data System (ADS)

    Borisov, A. Yu.

    2013-02-01

    A new method for estimation of the internal permittivity of photosynthetic membranes is based on joint analysis of the optical data with high spectral resolution and precise X-ray data. The permittivity of the bacteriochlorophyll-containing membranes of purple bacteria ranges from 1.62 to 1.75. The relatively low permittivity of photosynthetic organisms provides a significant increase in the efficiency of energy migration from multiple antenna chlorophylls to reaction centers and photosynthetic efficiency in general.

  7. Redox regulation of photosynthetic gene expression

    PubMed Central

    Queval, Guillaume; Foyer, Christine H.

    2012-01-01

    Redox chemistry and redox regulation are central to the operation of photosynthesis and respiration. However, the roles of different oxidants and antioxidants in the regulation of photosynthetic or respiratory gene expression remain poorly understood. Leaf transcriptome profiles of a range of Arabidopsis thaliana genotypes that are deficient in either hydrogen peroxide processing enzymes or in low molecular weight antioxidant were therefore compared to determine how different antioxidant systems that process hydrogen peroxide influence transcripts encoding proteins targeted to the chloroplasts or mitochondria. Less than 10 per cent overlap was observed in the transcriptome patterns of leaves that are deficient in either photorespiratory (catalase (cat)2) or chloroplastic (thylakoid ascorbate peroxidase (tapx)) hydrogen peroxide processing. Transcripts encoding photosystem II (PSII) repair cycle components were lower in glutathione-deficient leaves, as were the thylakoid NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) dehydrogenases (NDH) mRNAs. Some thylakoid NDH mRNAs were also less abundant in tAPX-deficient and ascorbate-deficient leaves. Transcripts encoding the external and internal respiratory NDHs were increased by low glutathione and low ascorbate. Regulation of transcripts encoding specific components of the photosynthetic and respiratory electron transport chains by hydrogen peroxide, ascorbate and glutathione may serve to balance non-cyclic and cyclic electron flow pathways in relation to oxidant production and reductant availability. PMID:23148274

  8. [Multiparticle computer simulation of protein interactions in the photosynthetic membrane].

    PubMed

    Riznichenko, G Iu; Kovalenko, I B; Abaturova, A M; D'iakonova, A N; Kniazeva, O S; Ustinin, D M; Khrushchev, S S; Rubin, A B

    2011-01-01

    The basic principles of the design of direct multiparticle models and the results of multiparticle computer simulation of electron transfer by mobile protein carriers in the photosynthetic membrane of a chloroplast thylakoid are presented. The reactions of complex formation of the protein plastocyanin with the protein cytochrome f and the pigment-protein complex of photosystem I, as well as of the protein ferredoxin with the protein FNR and photosystem 1 are considered. The role of diffusion and electrostatic interactions is discussed, and the effect of the shape of the reaction volume and ionic strength on the rate of electron transport are discussed. PMID:22117434

  9. Elementary Energy Transfer Pathways in Allochromatium vinosum Photosynthetic Membranes.

    PubMed

    Lüer, Larry; Carey, Anne-Marie; Henry, Sarah; Maiuri, Margherita; Hacking, Kirsty; Polli, Dario; Cerullo, Giulio; Cogdell, Richard J

    2015-11-01

    Allochromatium vinosum (formerly Chromatium vinosum) purple bacteria are known to adapt their light-harvesting strategy during growth according to environmental factors such as temperature and average light intensity. Under low light illumination or low ambient temperature conditions, most of the LH2 complexes in the photosynthetic membranes form a B820 exciton with reduced spectral overlap with LH1. To elucidate the reason for this light and temperature adaptation of the LH2 electronic structure, we performed broadband femtosecond transient absorption spectroscopy as a function of excitation wavelength in A. vinosum membranes. A target analysis of the acquired data yielded individual rate constants for all relevant elementary energy transfer (ET) processes. We found that the ET dynamics in high-light-grown membranes was well described by a homogeneous model, with forward and backward rate constants independent of the pump wavelength. Thus, the overall B800→B850→B890→ Reaction Center ET cascade is well described by simple triexponential kinetics. In the low-light-grown membranes, we found that the elementary backward transfer rate constant from B890 to B820 was strongly reduced compared with the corresponding constant from B890 to B850 in high-light-grown samples. The ET dynamics of low-light-grown membranes was strongly dependent on the pump wavelength, clearly showing that the excitation memory is not lost throughout the exciton lifetime. The observed pump energy dependence of the forward and backward ET rate constants suggests exciton diffusion via B850→ B850 transfer steps, making the overall ET dynamics nonexponential. Our results show that disorder plays a crucial role in our understanding of low-light adaptation in A. vinosum. PMID:26536265

  10. Effect of Chlamydomonas plastid terminal oxidase 1 expressed in tobacco on photosynthetic electron transfer.

    PubMed

    Feilke, Kathleen; Streb, Peter; Cornic, Gabriel; Perreau, François; Kruk, Jerzy; Krieger-Liszkay, Anja

    2016-01-01

    The plastid terminal oxidase PTOX is a plastohydroquinone:oxygen oxidoreductase that is important for carotenoid biosynthesis and plastid development. Its role in photosynthesis is controversially discussed. Under a number of abiotic stress conditions, the protein level of PTOX increases. PTOX is thought to act as a safety valve under high light protecting the photosynthetic apparatus against photodamage. However, transformants with high PTOX level were reported to suffer from photoinhibition. To analyze the effect of PTOX on the photosynthetic electron transport, tobacco expressing PTOX-1 from Chlamydomonas reinhardtii (Cr-PTOX1) was studied by chlorophyll fluorescence, thermoluminescence, P700 absorption kinetics and CO2 assimilation. Cr-PTOX1 was shown to compete very efficiently with the photosynthetic electron transport for PQH2 . High pressure liquid chromatography (HPLC) analysis confirmed that the PQ pool was highly oxidized in the transformant. Immunoblots showed that, in the wild-type, PTOX was associated with the thylakoid membrane only at a relatively alkaline pH value while it was detached from the membrane at neutral pH. We present a model proposing that PTOX associates with the membrane and oxidizes PQH2 only when the oxidation of PQH2 by the cytochrome b6 f complex is limiting forward electron transport due to a high proton gradient across the thylakoid membrane. PMID:26663146

  11. VIPP1 Has a Disordered C-Terminal Tail Necessary for Protecting Photosynthetic Membranes against Stress1[OPEN

    PubMed Central

    Zhang, Lingang; Kondo, Hideki

    2016-01-01

    Integrity of biomembranes is vital to living organisms. In bacteria, PspA is considered to act as repairing damaged membrane by forming large supercomplexes in Arabidopsis (Arabidopsis thaliana). Vulnerable to oxidative stress, photosynthetic organisms also contain a PspA ortholog called VIPP1, which has an additional C-terminal tail (Vc). In this study, Vc was shown to coincide with an intrinsically disordered region, and the role of VIPP1 in membrane protection against stress was investigated. We visualized VIPP1 by fusing it to GFP (VIPP1-GFP that fully complemented lethal vipp1 mutations), and investigated its behavior in vivo with live imaging. The intrinsically disordered nature of Vc enabled VIPP1 to form what appeared to be functional particles along envelopes, whereas the deletion of Vc caused excessive association of the VIPP1 particles, preventing their active movement for membrane protection. Expression of VIPP1 lacking Vc complemented vipp1 mutation, but exhibited sensitivity to heat shock stress. Conversely, transgenic plants over-expressing VIPP1 showed enhanced tolerance against heat shock, suggesting that Vc negatively regulates VIPP1 particle association and acts in maintaining membrane integrity. Our data thus indicate that VIPP1 is involved in the maintenance of photosynthetic membranes. During evolution, chloroplasts have acquired enhanced tolerance against membrane stress by incorporating a disordered C-terminal tail into VIPP1. PMID:27208228

  12. Abiotic Stresses: Insight into Gene Regulation and Protein Expression in Photosynthetic Pathways of Plants.

    PubMed

    Nouri, Mohammad-Zaman; Moumeni, Ali; Komatsu, Setsuko

    2015-01-01

    Global warming and climate change intensified the occurrence and severity of abiotic stresses that seriously affect the growth and development of plants,especially, plant photosynthesis. The direct impact of abiotic stress on the activity of photosynthesis is disruption of all photosynthesis components such as photosystem I and II, electron transport, carbon fixation, ATP generating system and stomatal conductance. The photosynthetic system of plants reacts to the stress differently, according to the plant type, photosynthetic systems (C₃ or C₄), type of the stress, time and duration of the occurrence and several other factors. The plant responds to the stresses by a coordinate chloroplast and nuclear gene expression. Chloroplast, thylakoid membrane, and nucleus are the main targets of regulated proteins and metabolites associated with photosynthetic pathways. Rapid responses of plant cell metabolism and adaptation to photosynthetic machinery are key factors for survival of plants in a fluctuating environment. This review gives a comprehensive view of photosynthesis-related alterations at the gene and protein levels for plant adaptation or reaction in response to abiotic stress. PMID:26343644

  13. Abiotic Stresses: Insight into Gene Regulation and Protein Expression in Photosynthetic Pathways of Plants

    PubMed Central

    Nouri, Mohammad-Zaman; Moumeni, Ali; Komatsu, Setsuko

    2015-01-01

    Global warming and climate change intensified the occurrence and severity of abiotic stresses that seriously affect the growth and development of plants, especially, plant photosynthesis. The direct impact of abiotic stress on the activity of photosynthesis is disruption of all photosynthesis components such as photosystem I and II, electron transport, carbon fixation, ATP generating system and stomatal conductance. The photosynthetic system of plants reacts to the stress differently, according to the plant type, photosynthetic systems (C3 or C4), type of the stress, time and duration of the occurrence and several other factors. The plant responds to the stresses by a coordinate chloroplast and nuclear gene expression. Chloroplast, thylakoid membrane, and nucleus are the main targets of regulated proteins and metabolites associated with photosynthetic pathways. Rapid responses of plant cell metabolism and adaptation to photosynthetic machinery are key factors for survival of plants in a fluctuating environment. This review gives a comprehensive view of photosynthesis-related alterations at the gene and protein levels for plant adaptation or reaction in response to abiotic stress. PMID:26343644

  14. Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: Identification of New Proteins

    SciTech Connect

    Zeng, Xiaohua; Roh, Jung Hyeob; Callister, Stephen J.; Tavano, Christine; Donohue, Timothy; Lipton, Mary S.; Kaplan, Samuel

    2007-10-01

    The intracytoplasmic membrane (ICM) system develops, upon induction, as a structure dedicated to the major events of bacterial photosynthesis, including harvesting light energy, primary charge separation, and electron transport. In this study, multi-chromatographic methods coupled with fourier transform ion cyclotron resonance (FTICR) mass spectrometer, combined with subcellular fractionation, was applied to an investigation of the supramolecular composition of the native photosynthetic membrane of Rhodobacter sphaeroides 2.4.1. A complete proteomic profile of the intracytoplasmic membranes was obtained and the results showed that the intracytoplasmic membranes are mainly composed of four photosynthetic membrane protein complexes, including light harvesting complexes I and II, the reaction center and cytochrome bc1, as well as two new membrane protein components, an unknown protein (RSP1760) and a possible alkane hydroxylase. Proteins necessary for various cellular functions, such as ATP synthesis, respiratory components, ABC transporters, protein translocation, and other proteins with unknown functions were also identified in association with the intracytoplasmic membranes. This study opens a new perspective on the characterization and understanding of the photosynthetic supramolecular complexes of R. sphaeroides, and their internal interactions as well as interactions with other proteins inside or outside the intracytoplasmic membranes.

  15. Photosynthetic bacteria production from food processing wastewater in sequencing batch and membrane photo-bioreactors.

    PubMed

    Chitapornpan, S; Chiemchaisri, C; Chiemchaisri, W; Honda, R; Yamamoto, K

    2012-01-01

    Application of photosynthetic process could be highly efficient and surpass anaerobic treatment in releasing less greenhouse gas and odor while the biomass produced can be utilized. The combination of photosynthetic process with membrane separation is possibly effective for water reclamation and biomass production. In this study, cultivation of mixed culture photosynthetic bacteria from food processing wastewater was investigated in a sequencing batch reactor (SBR) and a membrane bioreactor (MBR) supplied with infrared light. Both photo-bioreactors were operated at a hydraulic retention time (HRT) of 10 days. Higher MLSS concentration achieved in the MBR through complete retention of biomass resulted in a slightly improved performance. When the system was operated with MLSS controlled by occasional sludge withdrawal, total biomass production of MBR and SBR photo-bioreactor was almost equal. However, 64.5% of total biomass production was washed out with the effluent in SBR system. Consequently, the higher biomass could be recovered for utilization in MBR. PMID:22258682

  16. Visualizing the dynamic structure of the plant photosynthetic membrane.

    PubMed

    Ruban, Alexander V; Johnson, Matthew P

    2015-01-01

    The chloroplast thylakoid membrane is the site for the initial steps of photosynthesis that convert solar energy into chemical energy, ultimately powering almost all life on earth. The heterogeneous distribution of protein complexes within the membrane gives rise to an intricate three-dimensional structure that is nonetheless extremely dynamic on a timescale of seconds to minutes. These dynamics form the basis for the regulation of photosynthesis, and therefore the adaptability of plants to different environments. High-resolution microscopy has in recent years begun to provide new insights into the structural dynamics underlying a number of regulatory processes such as membrane stacking, photosystem II repair, photoprotective energy dissipation, state transitions and alternative electron transfer. Here we provide an overview of the essentials of thylakoid membrane structure in plants, and consider how recent advances, using a range of microscopies, have substantially increased our knowledge of the thylakoid dynamic structure. We discuss both the successes and limitations of the currently available techniques and highlight newly emerging microscopic methods that promise to move the field beyond the current 'static' view of membrane organization based on frozen snapshots to a 'live' view of functional membranes imaged under native aqueous conditions at ambient temperature and responding dynamically to external stimuli. PMID:27251532

  17. [Membrane-based photochemical systems as models for photosynthetic cells

    SciTech Connect

    Hurst, J.K.

    1992-01-01

    The objectives of this research are to improve our conceptual view of the ways in which membranes and interfaces can be used to control chemical reactivity. We have focused on understanding three elementary processes that are central to developing membrane-based integrated chemical systems for water photolysis or related photoconversion/photostorage processes. Specifically, we have sought to identify: the influence of interfaces upon charge separation/recombination reactions, pathways for transmembrane charge separation across hydrocarbon bilayer membranes, and mechanisms of water oxidation catalyzed by transition metal coordination complexes. Historically, the chemical dynamics of each of these processes has been poorly understood, with numerous unresolved issues and conflicting viewpoints appearing in the literature. As described in this report our recent research has led to considerable clarification of the underlying reaction mechanisms.

  18. Methods and constructs for expression of foreign proteins in photosynthetic organisms

    DOEpatents

    Laible, Philip D.; Hanson, Deborah K.

    2002-01-01

    A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

  19. Simple Colorimetric Determination of the Manganese Content in Photosynthetic Membranes

    SciTech Connect

    Semin, B. K.; Seibert, M.

    2009-01-01

    The functional Mn content of intact photosystem II membrane fragments was measured as 4.06 {+-} 0.13 Mn/reaction center when determined using a simple, sensitive colorimetric assay that will also work with thylakoids and core complexes. This procedure requires minimal sample material, does not need expensive assay equipment, requires four simple steps, and only takes 20-30 min to perform. These include (a) removal of the adventitious Mn ions by CaCl{sub 2} treatment of the membranes, (b) extraction of the Mn from the O{sub 2}-evolving complex with hydrochloric acid, (c) purification of the extract by centrifugation followed by filtration of the supernatant through an Acrodisc syringe filter (0.2 {micro}m nylon membrane), and (d) colorimetric determination of Mn in the extract using the reaction of the chromogenic agent, 3,3',5,5'-tetramethylbenzidine, with previously oxidized Mn(II) cations carried out at high pH. The colorimetric assay itself has been used previously by Serrat (Mikrochim Acta 129:77-80, 1998) for assaying Mn concentrations in sea water and drinking water.

  20. Kinetics of structural reorganizations in multilamellar photosynthetic membranes monitored by small-angle neutron scattering.

    PubMed

    Nagy, Gergely; Kovács, László; Ünnep, Renáta; Zsiros, Ottó; Almásy, László; Rosta, László; Timmins, Peter; Peters, Judith; Posselt, Dorthe; Garab, Győző

    2013-07-01

    We demonstrate the power of time-resolved small-angle neutron scattering experiments for the investigation of the structure and structural reorganizations of multilamellar photosynthetic membranes. In addition to briefly summarizing our results on thylakoid membranes isolated from higher plants and in unicellular organisms, we discuss the advantages and technical and methodological limitations of time-resolved SANS. We present a detailed and more systematical investigation of the kinetics of light-induced structural reorganizations in isolated spinach thylakoid membranes, which show how changes in the repeat distance and in the long-range order of the multilamellar membranes can be followed with a time resolution of seconds. We also present data from comparative measurements performed on thylakoid membranes isolated from tobacco. PMID:23839900

  1. Entropy and biological systems: Experimentally-investigated entropy-driven stacking of plant photosynthetic membranes

    NASA Astrophysics Data System (ADS)

    Jia, Husen; Liggins, John R.; Chow, Wah Soon

    2014-02-01

    According to the Second Law of Thermodynamics, an overall increase of entropy contributes to the driving force for any physicochemical process, but entropy has seldom been investigated in biological systems. Here, for the first time, we apply Isothermal Titration Calorimetry (ITC) to investigate the Mg2+-induced spontaneous stacking of photosynthetic membranes isolated from spinach leaves. After subtracting a large endothermic interaction of MgCl2 with membranes, unrelated to stacking, we demonstrate that the enthalpy change (heat change at constant pressure) is zero or marginally positive or negative. This first direct experimental evidence strongly suggests that an entropy increase significantly drives membrane stacking in this ordered biological structure. Possible mechanisms for the entropy increase include: (i) the attraction between discrete oppositely-charged areas, releasing counterions; (ii) the release of loosely-bound water molecules from the inter-membrane gap; (iii) the increased orientational freedom of previously-aligned water dipoles; and (iv) the lateral rearrangement of membrane components.

  2. Energy transfer and clustering of photosynthetic light-harvesting complexes in reconstituted lipid membranes

    NASA Astrophysics Data System (ADS)

    Dewa, Takehisa; Sumino, Ayumi; Watanabe, Natsuko; Noji, Tomoyasu; Nango, Mamoru

    2013-06-01

    In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light harvesting/reaction centre core complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. We addressed this issue by evaluating the energy transfer in reconstituted photosynthetic protein complexes LH2 and LH1-RC and studying the structures and the membrane environment of the LH2/LH1-RC assemblies, which had been embedded into various lipid bilayers. Thus, LH2 and LH1-RC from Rhodopseudomonas palustris 2.1.6 were reconstituted in phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE)/PG/cardiolipin (CL). Efficient energy transfer from LH2 to LH1-RC was observed in the PC and PE/PG/CL membranes. Atomic force microscopy revealed that LH2 and LH1-RC were heterogeneously distributed to form clusters in the PC and PE/PG/CL membranes. The results indicated that the phospholipid species influenced the cluster formation of LH2 and LH1-RC as well as the energy transfer efficiency.

  3. Conservation of distantly related membrane proteins: photosynthetic reaction centers share a common structural core.

    PubMed

    Sadekar, Sumedha; Raymond, Jason; Blankenship, Robert E

    2006-11-01

    Photosynthesis was established on Earth more than 3 billion years ago. All available evidences suggest that the earliest photosynthetic organisms were anoxygenic and that oxygen-evolving photosynthesis is a more recent development. The reaction center complexes that form the heart of the energy storage process are integral membrane pigment proteins that span the membrane in vectorial fashion to carry out electron transfer. The origin and extent of distribution of these proteins has been perplexing from a phylogenetic point of view mostly because of extreme sequence divergence. A series of integral membrane proteins of known structure and varying degrees of sequence identity have been compared using combinatorial extension-Monte Carlo methods. The proteins include photosynthetic reaction centers from proteobacteria and cyanobacterial photosystems I and II, as well as cytochrome oxidase, bacteriorhodopsin, and cytochrome b. The reaction center complexes show a remarkable conservation of the core structure of 5 transmembrane helices, strongly implying common ancestry, even though the residual sequence identity is less than 10%, whereas the other proteins have structures that are unrelated. A relationship of sequence with structure was derived from the reaction center structures; with characteristic decay length of 1.6 A. Phylogenetic trees derived from the structural alignments give insights into the earliest photosynthetic reaction center, strongly suggesting that it was a homodimeric complex that did not evolve oxygen. PMID:16887904

  4. Photoproduction of hydrogen by membranes of green photosynthetic bacteria

    SciTech Connect

    Bernstein, J D; Olson, J M

    1980-01-01

    Photoproduction of H/sub 2/ from ascorbate by unit-membrane vesicles from Chlorobium limicola f. thiosulfatophilum was achieved with a system containing gramicidin D, tetramethyl-p-phenylenediamine, methyl viologen, dithioerythritol, Clostridium hydrogenase, and an oxygen-scavenging mixture of glucose, glucose oxidase, ethanol, and catalase. Maximum quantum yield was less than one percent. Half maximum rate of H/sub 2/ production occurred at a white-light intensity of approximately 0.15 cm/sup -2/. The reaction was inhibited completely by 0.3% sodium dodecylbenzene sulfonate, 1% Triton X-100, or preheating the vesicles at 100/sup 0/C for 5 minutes. Low concentrations (0.01 and 0.05%) of Triton X-100 about doubled the reaction rate.

  5. Data supporting the absence of FNR dynamic photosynthetic membrane recruitment in trol mutants

    PubMed Central

    Vojta, Lea; Fulgosi, Hrvoje

    2016-01-01

    In photosynthesis, the flavoenzyme ferredoxin:NADP+ oxidoreductase (FNR) catalyses the final electron transfer from ferredoxin to NADP+, which is considered as the main pathway of high-energy electron partitioning in chloroplasts (DOI: 10.1111/j.1365-313X.2009.03999.x[1], DOI: 10.1038/srep10085[2]). Different detergents and pH treatments of photosynthetic membranes isolated from the Arabidopsis wild-type (WT) and the loss-of-function mutants of the thylakoid rhodanase-like protein TROL (trol), pre-acclimated to either dark, growth-light, or high-light conditions, were used to probe the strength of FNR-membrane associations. Detergents β-DM (decyl-β-D-maltopyranoside) or β-DDM (n-dodecyl-β-D-maltopyranoside) were used to test the stability of FNR binding to the thylakoid membranes, and to assess different membrane domains containing FNR. Further, the extraction conditions mimicked pH status of chloroplast stroma during changing light regimes. Plants without TROL are incapable of the dynamic FNR recruitment to the photosynthetic membranes. PMID:26977444

  6. Data supporting the absence of FNR dynamic photosynthetic membrane recruitment in trol mutants.

    PubMed

    Vojta, Lea; Fulgosi, Hrvoje

    2016-06-01

    In photosynthesis, the flavoenzyme ferredoxin:NADP(+) oxidoreductase (FNR) catalyses the final electron transfer from ferredoxin to NADP(+), which is considered as the main pathway of high-energy electron partitioning in chloroplasts (DOI: 10.1111/j.1365-313X.2009.03999.x[1], DOI: 10.1038/srep10085[2]). Different detergents and pH treatments of photosynthetic membranes isolated from the Arabidopsis wild-type (WT) and the loss-of-function mutants of the thylakoid rhodanase-like protein TROL (trol), pre-acclimated to either dark, growth-light, or high-light conditions, were used to probe the strength of FNR-membrane associations. Detergents β-DM (decyl-β-D-maltopyranoside) or β-DDM (n-dodecyl-β-D-maltopyranoside) were used to test the stability of FNR binding to the thylakoid membranes, and to assess different membrane domains containing FNR. Further, the extraction conditions mimicked pH status of chloroplast stroma during changing light regimes. Plants without TROL are incapable of the dynamic FNR recruitment to the photosynthetic membranes. PMID:26977444

  7. Interface for Light-Driven Electron Transfer by Photosynthetic Complexes Across Block Copolymer Membranes.

    PubMed

    Kuang, Liangju; Olson, Tien L; Lin, Su; Flores, Marco; Jiang, Yunjiang; Zheng, Wan; Williams, JoAnn C; Allen, James P; Liang, Hongjun

    2014-03-01

    Incorporation of membrane proteins into nanodevices to mediate recognition and transport in a collective and scalable fashion remains a challenging problem. We demonstrate how nanoscale photovoltaics could be designed using robust synthetic nanomembranes with incorporated photosynthetic reaction centers (RCs). Specifically, RCs from Rhodobacter sphaeroides are reconstituted spontaneously into rationally designed polybutadiene membranes to form hierarchically organized proteopolymer membrane arrays via a charge-interaction-directed reconstitution mechanism. Once incorporated, the RCs are fully active for prolonged periods based upon a variety of spectroscopic measurements, underscoring preservation of their 3D pigment configuration critical for light-driven charge transfer. This result provides a strategy to construct solar conversion devices using structurally versatile proteopolymer membranes with integrated RC functions to harvest broad regions of the solar spectrum. PMID:26274068

  8. Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes

    PubMed Central

    Sato, Norihiro; Hagio, Miki; Wada, Hajime; Tsuzuki, Mikio

    2000-01-01

    To investigate the role of phosphatidylglycerol (PG) in photosynthesis, we constructed a mutant defective in the CDP-diacylglycerol synthase gene from a cyanobacterium, Synechocystis sp. PCC6803. The mutant, designated as SNC1, required PG supplementation for growth. Growth was repressed in PG-free medium concomitantly with the decrease in cellular content of PG. These results indicate that PG is essential, and that SNC1 is defective in PG synthesis. Decrease in PG content was accompanied by a reduction in the cellular content of chlorophyll, but with little effect on the contents of phycobilisome pigments, which showed that levels of chlorophyll–protein complexes decreased without alteration of those of phycobilisomes. Regardless of the decrease in the PG content, CO2-dependent photosynthesis by SNC1 was similar to that by the wild type on a chlorophyll basis, but consequently became lower on a cell basis. Simultaneously, the ratio of oxygen evolution of photosystem II (PSII) measured with p-benzoquinone to that of CO2-dependent photosynthesis, which ranged between 1.3 and 1.7 in the wild type. However, it was decreased in SNC1 from 1.3 to 0.4 during the early growth phase where chlorophyll content and CO2-dependent photosynthesis were little affected, and then finally to 0.1, suggesting that PSII first lost its ability to reduce p-benzoquinone and then decreased in its level and actual activity. These results indicate that PG contributes to the accumulation of chlorophyll–protein complexes in thylakoid membranes, and also to normal functioning of PSII. PMID:10984546

  9. Quorum sensing influences growth and photosynthetic membrane production in high-cell-density cultivations of Rhodospirillum rubrum

    PubMed Central

    2013-01-01

    Background The facultative anoxygenic photosynthetic bacterium Rhodospirillum rubrum exhibits versatile metabolic activity allowing the adaptation to rapidly changing growth conditions in its natural habitat, the microaerobic and anoxic zones of stagnant waters. The microaerobic growth mode is of special interest as it allows the high-level expression of photosynthetic membranes when grown on succinate and fructose in the dark, which could significantly simplify the industrial production of compounds associated with PM formation. However, recently we showed that PM synthesis is no longer inducible when R. rubrum cultures are grown to high cell densities under aerobic conditions. In addition a reduction of the growth rate and the continued accumulation of precursor molecules for bacteriochlorophyll synthesis were observed under high cell densities conditions. Results In the present work, we demonstrate that the cell density-dependent effects are reversible if the culture supernatant is replaced by fresh medium. We identified six N-acylhomoserine lactones and show that four of them are produced in varying amounts according to the growth phase and the applied growth conditions. Further, we demonstrate that N-acylhomoserine lactones and tetrapyrrole compounds released into the growth medium affect the growth rate and PM expression in high cell density cultures. Conclusions In summary, we provide evidence that R. rubrum possesses a Lux-type quorum sensing system which influences the biosynthesis of PM and the growth rate and is thus likely to be involved in the phenotypes of high cell density cultures and the rapid adaptation to changing environmental conditions. PMID:23927486

  10. Effect of constitutive expression of bacterial phytoene desaturase CRTI on photosynthetic electron transport in Arabidopsis thaliana.

    PubMed

    Galzerano, Denise; Feilke, Kathleen; Schaub, Patrick; Beyer, Peter; Krieger-Liszkay, Anja

    2014-03-01

    The constitutive expression of the bacterial carotene desaturase (CRTI) in Arabidopsis thaliana leads to increased susceptibility of leaves to light-induced damage. Changes in the photosynthetic electron transport chain rather than alterations of the carotenoid composition in the antenna were responsible for the increased photoinhibition. A much higher level of superoxide/hydrogen peroxide was generated in the light in thylakoid membranes from the CRTI expressing lines than in wild-type while the level of singlet oxygen generation remained unchanged. The increase in reactive oxygen species was related to the activity of plastid terminal oxidase (PTOX) since their generation was inhibited by the PTOX-inhibitor octyl gallate, and since the protein level of PTOX was increased in the CRTI-expressing lines. Furthermore, cyclic electron flow was suppressed in these lines. We propose that PTOX competes efficiently with cyclic electron flow for plastoquinol in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool. PMID:24378845

  11. Differential stability of photosynthetic membranes and fatty acid composition at elevated temperature in Symbiodinium

    NASA Astrophysics Data System (ADS)

    Díaz-Almeyda, E.; Thomé, P. E.; El Hafidi, M.; Iglesias-Prieto, R.

    2011-03-01

    Coral reefs are threatened by increasing surface seawater temperatures resulting from climate change. Reef-building corals symbiotic with dinoflagellates in the genus Symbiodinium experience dramatic reductions in algal densities when exposed to temperatures above the long-term local summer average, leading to a phenomenon called coral bleaching. Although the temperature-dependent loss in photosynthetic function of the algal symbionts has been widely recognized as one of the early events leading to coral bleaching, there is considerable debate regarding the actual damage site. We have tested the relative thermal stability and composition of membranes in Symbiodinium exposed to high temperature. Our results show that melting curves of photosynthetic membranes from different symbiotic dinoflagellates substantiate a species-specific sensitivity to high temperature, while variations in fatty acid composition under high temperature rather suggest a complex process in which various modifications in lipid composition may be involved. Our results do not support the role of unsaturation of fatty acids of the thylakoid membrane as being mechanistically involved in bleaching nor as being a dependable tool for the diagnosis of thermal susceptibility of symbiotic reef corals.

  12. Gene expression responses in photosynthetic tissues to herbicides and pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    When plants are attacked by pathogens, the photosynthetic tissue is often dramatically affected. The chloroplasts within this tissue can participate in defense by being a source of many plant secondary metabolites that serve as defense signaling compounds, antioxidants, and phytoalexins. The chlorop...

  13. Spatial and temporal electroselection patterns in electric field stimulation of polarized luminescence from photosynthetic membrane vesicles

    PubMed Central

    Rosemberg, Yosef; Rozen, Philip; Malkin, Shmuel; Korenstein, Rafi

    1992-01-01

    Electroselection processes of charge recombination are manifested in the study of electric field induced polarized emission from photosynthetic membrane vesicles. The study explores the coupled spatial-temporal characteristics of electric field induced charge recombination by examining the dependence of the integrated polarized emission and the time dependent polarization on electric field strength. The experimental results were fitted to theoretical models by computer simulations employing empirical parameters. Simulation of the dependence of the integrated polarized components of emission on electric field strength, suggests field-dependent increased ratio between radiative and nonradiative rates of charge recombination. The observation that the initial polarization values are independent of electric field strength supports the assumption that electric field induced emission originates from the pole area and then spreads away from it towards the equator. The propagation rate of this electric field induced charge recombination from the pole area towards the equator is reflected by the decay of polarization which increases upon raising the electric field strength. Simulation of the polarization's decay, based on a calculated angle of 26.3 ± 0.4° between the transition moment of emission and the plane of the membrane, establishes coupled temporal spatial patterns of electroselection in intramembrane electron transfer invoked by exposing preilluminated photosynthetic vesicles to a homogeneous electric field. PMID:19431835

  14. Electric field-induced lateral mobility of photosystem I in the photosynthetic membrane

    PubMed Central

    Brumfeld, Vlad; Miller, Israel R.; Korenstein, Rafi

    1989-01-01

    Electrophoretic movement of photosystem I (PS I) along the photosynthetic membrane of hypotonically swollen thylakoid vesicles was studied by analyzing the electric field-stimulated delayed luminescence (electrophotoluminescence) emitted from PS I. The electrophoretic mobility was inferred from the differences in electrophotoluminescence (EPL) of the photosynthetic vesicles in presence and absence of trains of low amplitude (<80 V/cm) prepulses of 1 ms duration at 4 ms spacing. The average apparent electric mobility, determined from the time course of EPL increase on one hemisphere or its decrease on the other one, as function of prepulse length and intensity was of the order of 3 · 10-5 cm2V-1s-1. The assymetric distribution of the PS I reached a steady state when the diffusional, electrostatic, and elastic forces balanced the electrophoretic driving force. A lateral diffusion coefficient of ∼5 · 10-9 cm2s-1 was found for the PS I complex from the diffusional relaxation after cessation of the electric field pulse train. Experimental conditions such as concentration, temperature, and viscosity of the aqueous solution were not critical for the effect. Between 23 and 150 electron charges per moving particle were estimated from the measured electrophoretic mobility. PMID:19431746

  15. Characterization of a photosynthetic microbial solar cell with a membrane electrode assembly

    NASA Astrophysics Data System (ADS)

    Kim, Jin-Hee; Bai, Seoung Jai

    2014-07-01

    We studied the photosynthetic microbial solar cell (PMSC), which is one of the sources of renewable energy. In order to increase the power output of the PMSC, we fabricated a membrane electrode assembly (MEA) by either spin coating or hot pressing with a Pt catalytic layer, whose thickness was changed depending on the fabrication method. The PMSC with MEA fabricated by hot pressing with a thick Pt layer showed higher power output than that fabricated by spin coating with a thin Pt layer. The effectiveness of the fabrication method and the correlation between the Pt thickness and the maximum power output of the PMSC were explained by using the triphase boundary theory and were verified experimentally using energy dispersive X-ray spectrometry (EDS) line scans across the MEA cross sections.

  16. Effects of grafting on key photosynthetic enzymes and gene expression in the citrus cultivar Huangguogan.

    PubMed

    Liao, L; Cao, S Y; Rong, Y; Wang, Z H

    2016-01-01

    Grafting influences scion photosynthetic capacity and fruit quality. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which strongly affects photosynthetic rate, and Rubisco activase (RCA), which regulates Rubisco activity, are two key photosynthetic enzymes. However, little information is available regarding the effect of grafting on the concentration and expression of Rubisco and RCA in the citrus cultivar Huangguogan. The objective of this study was to investigate the effect of grafting Huangguogan plants onto trifoliate orange, tangerine, and orange on: 1) the concentration of Rubisco and RCA; 2) the mRNA levels of rbcL, rbcS, and rca; and 3) fruit quality. Overall, the results showed that when Huangguogan plants budded on tangerine and orange, they had better fruit quality, while on trifoliate orange they had higher Rubisco concentration. Tangerine and orange are probably the most suitable rootstocks for Huangguogan plants given the environmental conditions of Sichuan Province, China. PMID:26985941

  17. Membrane protein expression in Lactococcus lactis.

    PubMed

    King, Martin S; Boes, Christoph; Kunji, Edmund R S

    2015-01-01

    The Gram-positive bacterium Lactococcus lactis has many properties that are ideal for the overproduction of membrane proteins in a functional form. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. The available promoter systems are strong and tightly regulated, allowing expression of toxic gene products in a controlled manner. Expressed membrane proteins are targeted exclusively to the cytoplasmic membrane, allowing the use of ionophores, ligands, and inhibitors to study activity of the membrane protein in whole cells. Constructed plasmids are stable and expression levels are highly reproducible. The relatively small genome size of the organism causes little redundancy, which facilitates complementation studies and allows for easier purification. The produced membrane proteins are often stable, as the organism has limited proteolytic capability, and they are readily solubilized from the membrane with mild detergents. Lactococci are multiple amino acid auxotrophs, allowing the incorporation of labels, such as selenomethionine. Among the few disadvantages are the low transformation frequency, AT-rich codon usage, and resistance to lysis by mechanical means, but these problems can be overcome fairly easily. We will describe in detail the protocols used to express membrane proteins in L. lactis, from cloning of the target gene to the isolation of membrane vesicles for the determination of expression levels. PMID:25857778

  18. Orientation of chlorophylls within chloroplasts as shown by optical and electrochromic properties of the photosynthetic membrane.

    PubMed

    Paillotin, G; Breton, J

    1977-04-01

    The effects on the optical properties of photosynthetic membranes caused by several types of chlorophyll differing in resonance frequency and in spatial disposition are theoretically analyzed. Using a method of moments and the linear dichroism spectrum of the lamellae, we evaluated the mean angle (phi) between the transition moment of each chlorophyll and the normal to the lamellae. We have confirmed that at about 695 nm the transition moment is in the plane of the lamellae, and outside it for chlorophyll b (phi approximately 48.6 degrees). By integrating over frequency the absorption variations affected by ionophores, we show that they may be ascribed to a Stark effect, and we analyze the dependence of this effect on the orientation of the chlorophylls. From this dependence and the degree of polarization of the Stark effect, we calculate the spatial fluctuations of the angle phi. The calculation shows that a definite value of phi corresponds to each resonance frequency of chlorophyl a found in vivo. This proves that the chlorophylls a are not oriented partly random. For chlorophylls b, on the other hand, phi may fluctuate by some 10 degrees about its mean value. The structural consequences of these results are discussed. PMID:851575

  19. Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae

    DOEpatents

    Melis, Anastasios; Mitra, Mautusi

    2010-06-29

    The invention provides method and compositions to minimize the chlorophyll antenna size of photosynthesis by decreasing TLA1 gene expression, thereby improving solar conversion efficiencies and photosynthetic productivity in plants, e.g., green microalgae, under bright sunlight conditions.

  20. Heterologous expression of Arabidopsis phytochrome B in transgenic potato influences photosynthetic performance and tuber development

    SciTech Connect

    Thiele, A.; Herold, M.; Lenk, I.; Gatz, C. . Albrecht von Haller Inst. fuer Pflanzenwissenschaften); Quail, P.H. )

    1999-05-01

    Transgenic potato (Solanum tuberosum) plants expressing Arabidopsis phytochrome B were characterized morphologically and physiologically under white light in a greenhouse to explore their potential for improved photosynthesis and higher tuber yields. As expected, overexpression of functional phytochrome B caused pleiotropic effects such as semidwarfism, decreased apical dominance, a higher number of smaller but thicker leaves, and increased pigmentation. Because of increased numbers of chloroplasts in elongated palisade cells, photosynthesis per leaf area and in each individual plant increased. In addition, photosynthesis was less sensitive to photoinactivation under prolonged light stress. The beginning of senescence was not delayed, but deceleration of chlorophyll degradation extended the lifetime of photosynthetically active plants. Both the higher photosynthetic performance and the longer lifespan of the transgenic plants allowed greater biomass production, resulting in extended underground organs with increased tuber yields.

  1. THE STATE OF MANGANESE IN THE PHOTOSYNTHETIC APPARATUS. II. X-RAY ABSORPTION EDGE STUDIES ON MANGANESE IN PHOTOSYNTHETIC MEMBRANES

    SciTech Connect

    Kirby, J. A.; Goodin, D. B.; Wydrzynski, T.; Robertson, A. S.; Klein, M. P.

    1980-11-01

    X-ray absorption spectra at the Manganese K-edge are presented for spinach chloroplasts, and chloroplasts which have been Tris-treated and hence unable to evolve oxygen. A significant change in the electronic environment of manganese is observed and is attributed to the release of manganese from the thylakoid membranes with a concomitant change in oxidation state. A correlation of the K-edge energy, defined as the energy at the first inflection point, with coordination charge has been established for a number of manganese compounds of known structure and oxidation state. Comparison of the manganese K-edge energies of the chloroplast samples with the reference compounds places the average oxidation state of the chloroplasts between +2 and +3. Using the edge spectra for Tris-treated membranes which were osmotically shocked to remove the released manganese, difference edge spectra were synthesized to approximate the active pool of manganese. Coordination charge predictions for this fraction are consistent with an average resting oxidation state higher than +2. The shape at the edge is also indicative of heterogeneity of the manganese site, of low symmetry, or both.

  2. Organic carbon recovery and photosynthetic bacteria population in an anaerobic membrane photo-bioreactor treating food processing wastewater.

    PubMed

    Chitapornpan, S; Chiemchaisri, C; Chiemchaisri, W; Honda, R; Yamamoto, K

    2013-08-01

    Purple non-sulfur bacteria (PNSB) were cultivated by food industry wastewater in the anaerobic membrane photo-bioreactor. Organic removal and biomass production and characteristics were accomplished via an explicit examination of the long term performance of the photo-bioreactor fed with real wastewater. With the support of infra-red light transmitting filter, PNSB could survive and maintain in the system even under the continual fluctuations of influent wastewater characteristics. The average BOD and COD removal efficiencies were found at the moderate range of 51% and 58%, respectively. Observed photosynthetic biomass yield was 0.6g dried solid/g BOD with crude protein content of 0.41 g/g dried solid. Denaturing gradient gel electrophoretic analysis (DGGE) and 16S rDNA sequencing revealed the presence of Rhodopseudomonas palustris and significant changes in the photosynthetic bacterial community within the system. PMID:23489563

  3. Unique role for translation initiation factor 3 in the light color regulation of photosynthetic gene expression.

    PubMed

    Gutu, Andrian; Nesbit, April D; Alverson, Andrew J; Palmer, Jeffrey D; Kehoe, David M

    2013-10-01

    Light-harvesting antennae are critical for collecting energy from sunlight and providing it to photosynthetic reaction centers. Their abundance and composition are tightly regulated to maintain efficient photosynthesis in changing light conditions. Many cyanobacteria alter their light-harvesting antennae in response to changes in ambient light-color conditions through the process of chromatic acclimation. The control of green light induction (Cgi) pathway is a light-color-sensing system that controls the expression of photosynthetic genes during chromatic acclimation, and while some evidence suggests that it operates via transcription attenuation, the components of this pathway have not been identified. We provide evidence that translation initiation factor 3 (IF3), an essential component of the prokaryotic translation initiation machinery that binds the 30S subunit and blocks premature association with the 50S subunit, is part of the control of green light induction pathway. Light regulation of gene expression has not been previously described for any translation initiation factor. Surprisingly, deletion of the IF3-encoding gene infCa was not lethal in the filamentous cyanobacterium Fremyella diplosiphon, and its genome was found to contain a second, redundant, highly divergent infC gene which, when deleted, had no effect on photosynthetic gene expression. Either gene could complement an Escherichia coli infC mutant and thus both encode bona fide IF3s. Analysis of prokaryotic and eukaryotic genome databases established that multiple infC genes are present in the genomes of diverse groups of bacteria and land plants, most of which do not undergo chromatic acclimation. This suggests that IF3 may have repeatedly evolved important roles in the regulation of gene expression in both prokaryotes and eukaryotes. PMID:24048028

  4. Analysis of picosecond laser induced fluorescence phenomena in photosynthetic membranes utilizing a master equation approach.

    PubMed Central

    Paillotin, G; Swenberg, C E; Breton, J; Geacintov, N E

    1979-01-01

    A Pauli master equation is formulated and solved to describe the fluorescence quantum yield, phi, and the fluorescence temporal decay curves. F(t), obtained in picosecond laser excitation experiments of photosynthetic systems. It is assumed that the lowering of phi with increasing pulse intensity is due to bimolecular singlet exciton annihilation processes which compete with the monomolecular exciton decay processes; Poisson statistics are taken into account. Calculated curves of phi as a function of the number of photon hits per domain are compared with experimental data, and it is concluded that these domains contain at least two to four connected photosynthetic units (depending on the temperature), where each photosynthetic unit is assumed to contain approximately 300 pigment molecules. It is shown that under conditions of high excitation intensities, the fluorescence decays approximately according to the (time)1/2 law. PMID:262402

  5. Analysis of picosecond laser induced fluorescence phenomena in photosynthetic membranes utilizing a master equation approach.

    PubMed

    Paillotin, G; Swenberg, C E; Breton, J; Geacintov, N E

    1979-03-01

    A Pauli master equation is formulated and solved to describe the fluorescence quantum yield, phi, and the fluorescence temporal decay curves. F(t), obtained in picosecond laser excitation experiments of photosynthetic systems. It is assumed that the lowering of phi with increasing pulse intensity is due to bimolecular singlet exciton annihilation processes which compete with the monomolecular exciton decay processes; Poisson statistics are taken into account. Calculated curves of phi as a function of the number of photon hits per domain are compared with experimental data, and it is concluded that these domains contain at least two to four connected photosynthetic units (depending on the temperature), where each photosynthetic unit is assumed to contain approximately 300 pigment molecules. It is shown that under conditions of high excitation intensities, the fluorescence decays approximately according to the (time)1/2 law. PMID:262402

  6. Distribution and self-organization of photosynthetic pigments in micelles: implication for the assembly of light-harvesting complexes and reaction centers in the photosynthetic membrane.

    PubMed Central

    Scherz, A; Rosenbach-Belkin, V; Fisher, J R

    1990-01-01

    The addition of bacteriochlorophylls and bacteriopheophytins to formamide/water, 3:1 (vol/vol), (or water) containing small spherical micelles of Triton X-100 leads to the reorganization of the detergent into micelles that consist of 5000-40,000 amphiphilic molecules. The pigment distribution within the micelles was determined by modified Poisson statistics taking into consideration the various sizes of micelles. Pigment dimerization occurred in micelles with more than a single occupant and was driven by a free-energy change of -4.5 kcal/mol (1 cal = 4.184 J) for bacteriochlorophyll a in formamide/water, -7.6 kcal/mol for bacteriopheophytin a in formamide/water, and -6.6 kcal/mol for bacteriopheophytin a in water. These values correspond to the room temperature equilibrium constants 2.2 x 10(3) M-1, 3.9 x 10(5) M-1, and 7.5 x 10(4) M-1, respectively. The incorporation of bacteriochlorophylls with attached small formamide polymers and the subsequent dimerization of these pigments in the lipid phase provide a model for studying the synergetic organization of polypeptides and bacteriochlorophyll clusters in the photosynthetic membrane. PMID:11607092

  7. Molecular cloning of a maize gene involved in photosynthetic membrane organization that is regulated by Robertson's Mutator.

    PubMed Central

    Martienssen, R A; Barkan, A; Freeling, M; Taylor, W C

    1989-01-01

    The maize photosynthetic mutant hcf106 has a distinctive and unusual thylakoid membrane organization, and fails to accumulate three of the five thylakoid membrane protein complexes. This mutant arose in a Robertson's Mutator background, and shows somatic instability typical of a transposon-induced allele. In addition, hcf106 is suppressed when Mu1 elements are inactive and modified in their terminal inverted repeats. Thus plants homozygous for the mutant allele adopt a mutant phenotype only when Mu1 elements are active and unmodified. DNA from the mutant allele has been cloned by 'transposon-tagging' using the transposon Mu1, and the identity of the clone confirmed by observing somatic excision of the transposon in a revertant sector. A 1.2 kb transcript homologous to the cloned DNA is found in wild-type and suppressed seedlings, but is not found in mutant seedlings, suggesting that suppression is mediated at the level of transcript accumulation. Images PMID:2548850

  8. Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii.

    PubMed

    Hu, Zhangli; Zhao, Zhonglin; Wu, Zhihua; Fan, Zhun; Chen, Jun; Wu, Jinxia; Li, Jiancheng

    2011-09-01

    The efficient expression of exogenous gene in mitochondria of photosynthetic organism has been an insurmountable problem. In this study, the pBsLPNCG was constructed by inserting the egfp gene into a site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA of Chlamydomonas reinhardtii CC-124 and introduced into the mitochondria of respiratory deficient dum-1 mutation of C. reinhardtii CC-2654. Sequencing and DNA Southern analyses revealed that egfp gene had been integrated into the mitochondrial genome of transgenic algae as expected and no other copy of egfp existed in their nucleic genome. Both the fluorescence detection and Western blot analysis confirmed the presence of eGFP protein in the transgenic algae; it indicated that the egfp gene was successfully expressed in the mitochondria of C. reinhardtii. PMID:21664493

  9. C4 Photosynthetic Gene Expression in Light- and Dark-Grown Amaranth Cotyledons.

    PubMed

    Wang, J. L.; Long, J. J.; Hotchkiss, T.; Berry, J. O.

    1993-08-01

    The patterns of expression for genes encoding several C4 photosynthetic enzymes were examined in light-grown and dark-grown (etiolated) cotyledons of amaranth (Amaranthus hypochondriacus), a dicotyledonous C4 plant. The large subunit and small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase), phosphoenolpyruvate carboxylase (PEPCase), and pyruvate orthophosphate dikinase (PPdK) were all present in the cotyledons by d 2 after planting when the seedlings first emerged from the seed coat. Kranz anatomy was apparent in light-grown cotyledons throughout development, and the overall patterns of C4 gene expression were similar to those recently described for developing amaranth leaves (J.L. Wang, D.F. Klessig, J.O. Berry [1992] Plant Cell 4: 173-184). RuBPCase mRNA and proteins were present in both bundle sheath and mesophyll cells in a C3-like pattern during early development and became progressively more localized to bundle sheath cells in the C4-type pattern as the cotyledons expanded over 2 to 7 d. PEPCase and PPdK polypeptides were localized to mesophyll cells throughout development, even though PEPCase transcripts were detected in both bundle sheath and mesophyll cells. Kranz anatomy also developed in cotyledons grown in complete darkness. In 7-d-old dark-grown cotyledons, RuBPCase, PPdK, and PEPCase were all localized to the appropriate cell types, although at somewhat lower levels than in light-grown cotyledons. These findings demonstrate that the leaves and postembryonic cotyledons of amaranth undergo common developmental programs of C4 gene expression during maturation. Furthermore, light is not required for the cell-type-specific expression of genes encoding RuBPCase and other photosynthetic enzymes in this dicotyledonous C4 plant. PMID:12231890

  10. Atomic Detail Visualization of Photosynthetic Membranes with GPU-Accelerated Ray Tracing

    PubMed Central

    Vandivort, Kirby L.; Barragan, Angela; Singharoy, Abhishek; Teo, Ivan; Ribeiro, João V.; Isralewitz, Barry; Liu, Bo; Goh, Boon Chong; Phillips, James C.; MacGregor-Chatwin, Craig; Johnson, Matthew P.; Kourkoutis, Lena F.; Hunter, C. Neil

    2016-01-01

    The cellular process responsible for providing energy for most life on Earth, namely photosynthetic light-harvesting, requires the cooperation of hundreds of proteins across an organelle, involving length and time scales spanning several orders of magnitude over quantum and classical regimes. Simulation and visualization of this fundamental energy conversion process pose many unique methodological and computational challenges. We present, in two accompanying movies, light-harvesting in the photosynthetic apparatus found in purple bacteria, the so-called chromatophore. The movies are the culmination of three decades of modeling efforts, featuring the collaboration of theoretical, experimental, and computational scientists. We describe the techniques that were used to build, simulate, analyze, and visualize the structures shown in the movies, and we highlight cases where scientific needs spurred the development of new parallel algorithms that efficiently harness GPU accelerators and petascale computers. PMID:27274603

  11. [Effects of exogenous salicylic acid on membrane lipid peroxidation and photosynthetic characteristics of Cucumis sativus seedlings under drought stress].

    PubMed

    Hao, Jing-Hong; Yi, Yang; Shang, Qing-Mao; Dong, Chun-Juan; Zhang, Zhi-Gang

    2012-03-01

    To approach the related mechanisms of exogenous salicylic acid (SA) in improving plant drought-resistance, this paper studied the effects of applying exogenous SA to the rhizosphere on the plant growth, membrane lipid peroxidation, proline accumulation, water use efficiency, net photosynthetic rate (Pn), and chlorophyll fluorescence parameters of cucumber (Cucumis sativus) seedlings under drought stresses (60% and 50% of saturated water capacity). Applying SA relieved the inhibitory effects of drought stress on plant growth, Pn, and water use efficiency, decreased membrane lipid peroxidation, and promoted proline accumulation. Meanwhile, the SA decreased the decrements of the maximum photochemical efficiency of PS II, actual photochemical efficiency of PS II, potential activity of PS II, effective photochemical efficiency of PS II, and photochemical quenching coefficient under drought stress significantly, and limited the increase of non-photochemical quenching coefficient. All the results suggested that applying exogenous SA could alleviate the oxidation damage of cell membrane resulted from the drought-caused membrane lipid peroxidation, improve the Pn by increasing PS II activity to benefit water utilization, enhance the regulation capability of osmosis to decrease water loss and increase water use efficiency, and thereby, improve the plant drought-resistance. PMID:22720616

  12. The electron conduction of photosynthetic protein complexes embedded in a membrane.

    PubMed

    Stamouli, A; Frenken, J W M; Oosterkamp, T H; Cogdell, R J; Aartsma, T J

    2004-02-27

    The conductivity of two photosynthetic protein-pigment complexes, a light harvesting 2 complex and a reaction center, was measured with an atomic force microscope capable of performing electrical measurements. Current-voltage measurements were performed on complexes embedded in their natural environment. Embedding the complexes in lipid bilayers made it possible to discuss the different conduction behaviors of the two complexes in light of their atomic structure. PMID:14988007

  13. 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

  14. Electric field-induced lateral mobility of photosystem I in the photosynthetic membrane: A study by electrophotoluminescence.

    PubMed

    Brumfeld, V; Miller, I R; Korenstein, R

    1989-09-01

    Electrophoretic movement of photosystem I (PS I) along the photosynthetic membrane of hypotonically swollen thylakoid vesicles was studied by analyzing the electric field-stimulated delayed luminescence (electrophotoluminescence) emitted from PS I. The electrophoretic mobility was inferred from the differences in electrophotoluminescence (EPL) of the photosynthetic vesicles in presence and absence of trains of low amplitude (<80 V/cm) prepulses of 1 ms duration at 4 ms spacing. The average apparent electric mobility, determined from the time course of EPL increase on one hemisphere or its decrease on the other one, as function of prepulse length and intensity was of the order of 3 . 10(-5) cm(2)V(-1)s(-1). The assymetric distribution of the PS I reached a steady state when the diffusional, electrostatic, and elastic forces balanced the electrophoretic driving force. A lateral diffusion coefficient of approximately 5 . 10(-9) cm(2)s(-1) was found for the PS I complex from the diffusional relaxation after cessation of the electric field pulse train. Experimental conditions such as concentration, temperature, and viscosity of the aqueous solution were not critical for the effect. Between 23 and 150 electron charges per moving particle were estimated from the measured electrophoretic mobility. PMID:19431746

  15. Kinetic development and evaluation of membrane sequencing batch reactor (MSBR) with mixed cultures photosynthetic bacteria for dairy wastewater treatment.

    PubMed

    Kaewsuk, Jutamas; Thorasampan, Worachat; Thanuttamavong, Monthon; Seo, Gyu Tae

    2010-05-01

    This experimental study was conducted to evaluate a membrane sequencing batch reactor (MSBR) with mixed culture photosynthetic bacteria for dairy wastewater treatment. The study was undertaken in two steps: laboratory and pilot scale experiments. In the first step, kinetics analysis of the MSBR was carried out in a laboratory scale experiment with influent COD concentration of 2500 mg/L. The pilot scale experiment was conducted to investigate the performance of the MSBR and checked the suitability of the kinetics for an engineering design. The kinetic coefficients K(s), k, k(d), Y and mu(m) were found to be 174-mg-COD/L, 7.42/d, 0.1383/d, 0.2281/d and 1.69/d, respectively. There were some deviations of COD removal efficiency between the design value and the actual value. From the kinetics estimation, COD effluent from the design was 27 mg/L while the average actual COD effluent from the experiment was 149 mg/L. Due to the different light source condition, the factors relating to light energy (i.e. L(f) and IR(%)) must be incorporated into engineering design and performance prediction with these kinetic coefficients of the photosynthetic MSBR. PMID:20149520

  16. Photosynthetic membrane-less microbial fuel cells to enhance microalgal biomass concentration.

    PubMed

    Uggetti, Enrica; Puigagut, Jaume

    2016-10-01

    The aim of this study was to quantitatively assess the net increase in microalgal biomass concentration induced by photosynthetic microbial fuel cells (PMFC). The experiment was conducted on six lab-scale PMFC constituted by an anodic chamber simulating an anaerobic digester connected to a cathodic chamber consisting of a mixed algae consortia culture. Three PMFC were operated at closed circuit (PMFC(+)) whereas three PMFC were left unconnected as control (PMFC(-)). PMFC(+) produced a higher amount of carbon dioxide as a product of the organic matter oxidation that resulted in 1.5-3 times higher biomass concentration at the cathode compartment when compared to PMFC(-). PMID:27455126

  17. Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy.

    PubMed

    Charuvi, Dana; Nevo, Reinat; Kaplan-Ashiri, Ifat; Shimoni, Eyal; Reich, Ziv

    2016-01-01

    Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (>100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ. PMID:27403565

  18. Ionic liquids effects on the permeability of photosynthetic membranes probed by the electrochromic shift of endogenous carotenoids.

    PubMed

    Malferrari, Marco; Malferrari, Danilo; Francia, Francesco; Galletti, Paola; Tagliavini, Emilio; Venturoli, Giovanni

    2015-11-01

    Ionic liquids (ILs) are promising materials exploited as solvents and media in many innovative applications, some already used at the industrial scale. The chemical structure and physicochemical properties of ILs can differ significantly according to the specific applications for which they have been synthesized. As a consequence, their interaction with biological entities and toxicity can vary substantially. To select highly effective and minimally harmful ILs, these properties need to be investigated. Here we use the so called chromatophores--protein-phospholipid membrane vesicles obtained from the photosynthetic bacterium Rhodobacter sphaeroides--to assess the effects of imidazolinium and pyrrolidinium ILs, with chloride or dicyanamide as counter anions, on the ionic permeability of a native biological membrane. The extent and modalities by which these ILs affect the ionic conductivity can be studied in chromatophores by analyzing the electrochromic response of endogenous carotenoids, acting as an intramembrane voltmeter at the molecular level. We show that chromatophores represent an in vitro experimental model suitable to probe permeability changes induced in cell membranes by ILs differing in chemical nature, degree of oxygenation of the cationic moiety and counter anion. PMID:26343161

  19. Identification of an Atypical Membrane Protein Involved in the Formation of Protein Disulfide Bonds in Oxygenic Photosynthetic Organisms*S⃞

    PubMed Central

    Singh, Abhay K.; Bhattacharyya-Pakrasi, Maitrayee; Pakrasi, Himadri B.

    2008-01-01

    The evolution of oxygenic photosynthesis in cyanobacteria nearly three billion years ago provided abundant reducing power and facilitated the elaboration of numerous oxygen-dependent reactions in our biosphere. Cyanobacteria contain an internal thylakoid membrane system, the site of photosynthesis, and a typical Gram-negative envelope membrane system. Like other organisms, the extracytoplasmic space in cyanobacteria houses numerous cysteine-containing proteins. However, the existence of a biochemical system for disulfide bond formation in cyanobacteria remains to be determined. Extracytoplasmic disulfide bond formation in non-photosynthetic organisms is catalyzed by coordinated interaction between two proteins, a disulfide carrier and a disulfide generator. Here we describe a novel gene, SyndsbAB, required for disulfide bond formation in the extracytoplasmic space of cyanobacteria. The SynDsbAB orthologs are present in most cyanobacteria and chloroplasts of higher plants with fully sequenced genomes. The SynDsbAB protein contains two distinct catalytic domains that display significant similarity to proteins involved in disulfide bond formation in Escherichia coli and eukaryotes. Importantly, SyndsbAB complements E. coli strains defective in disulfide bond formation. In addition, the activity of E. coli alkaline phosphatase localized to the periplasm of Synechocystis 6803 is dependent on the function of SynDsbAB. Deletion of SyndsbAB in Synechocystis 6803 causes significant growth impairment under photoautotrophic conditions and results in hyper-sensitivity to dithiothreitol, a reductant, whereas diamide, an oxidant had no effect on the growth of the mutant strains. We conclude that SynDsbAB is a critical protein for disulfide bond formation in oxygenic photosynthetic organisms and required for their optimal photoautotrophic growth. PMID:18413314

  20. Suppression of Photosynthetic Gene Expression in Roots Is Required for Sustained Root Growth under Phosphate Deficiency1[W][OPEN

    PubMed Central

    Kang, Jun; Yu, Haopeng; Tian, Caihuan; Zhou, Wenkun; Li, Chuanyou; Jiao, Yuling; Liu, Dong

    2014-01-01

    Plants cope with inorganic phosphate (Pi) deficiencies in their environment by adjusting their developmental programs and metabolic activities. For Arabidopsis (Arabidopsis thaliana), the developmental responses include the inhibition of primary root growth and the enhanced formation of lateral roots and root hairs. Pi deficiency also inhibits photosynthesis by suppressing the expression of photosynthetic genes. Early studies showed that photosynthetic gene expression was also suppressed in Pi-deficient roots, a nonphotosynthetic organ; however, the biological relevance of this phenomenon remains unknown. In this work, we characterized an Arabidopsis mutant, hypersensitive to Pi starvation7 (hps7), that is hypersensitive to Pi deficiency; the hypersensitivity includes an increased inhibition of root growth. HPS7 encodes a tyrosylprotein sulfotransferase. Accumulation of HPS7 proteins in root tips is enhanced by Pi deficiency. Comparative RNA sequencing analyses indicated that the expression of many photosynthetic genes is activated in roots of hps7. Under Pi deficiency, the expression of photosynthetic genes in hps7 is further increased, which leads to enhanced accumulation of chlorophyll, starch, and sucrose. Pi-deficient hps7 roots also produce a high level of reactive oxygen species. Previous research showed that the overexpression of GOLDEN-like (GLK) transcription factors in transgenic Arabidopsis activates photosynthesis in roots. The GLK overexpressing (GLK OX) lines also exhibit increased inhibition of root growth under Pi deficiency. The increased inhibition of root growth in hps7 and GLK OX lines by Pi deficiency was completely reversed by growing the plants in the dark. Based on these results, we propose that suppression of photosynthetic gene expression is required for sustained root growth under Pi deficiency. PMID:24868033

  1. Kinetic model of primary energy transfer and trapping in photosynthetic membranes

    PubMed Central

    Pullerits, Tõnu; Freiberg, Arvi

    1992-01-01

    The picosecond time-domain incoherent singlet excitation transfer and trapping kinetics in core antenna of photosynthetic bacteria are studied in case of low excitation intensities by numerical integration of the appropriate master equation in a wide temperature range of 4-300 K. The essential features of our two-dimensional-lattice model are as follows: Förster excitation transfer theory, spectral heterogeneity of both the light-harvesting antenna and the reaction center, treatment of temperature effects through temperature dependence of spectral bands, inclusion of inner structure of the trap, and transition dipole moment orientation. The fluorescence kinetics is analyzed in terms of distributions of various kinetic components, and the influence of different inhomogeneities (orientational, spectral) is studied. A reasonably good agreement between theoretical and experimental fluorescence decay kinetics for purple photosynthetic bacterium Rhodospirillum rubrum is achieved at high temperatures by assuming relatively large antenna spectral inhomogeneity: 20 nm at the whole bandwidth of 40 nm. The mean residence time in the antenna lattice site (it is assumed to be the aggregate of four bacteriochlorophyll a molecules bound to proteins) is estimated to be ∼12 ps. At 4 K only qualitative agreement between model and experiment is gained. The failure of quantitative fitting is perhaps due to the lack of knowledge about the real structure of antenna or local heating and cooling effects not taken into account. PMID:19431849

  2. Optimal fold symmetry of LH2 rings on a photosynthetic membrane

    PubMed Central

    Cleary, Liam; Chen, Hang; Chuang, Chern; Silbey, Robert J.; Cao, Jianshu

    2013-01-01

    An intriguing observation of photosynthetic light-harvesting systems is the N-fold symmetry of light-harvesting complex 2 (LH2) of purple bacteria. We calculate the optimal rotational configuration of N-fold rings on a hexagonal lattice and establish two related mechanisms for the promotion of maximum excitation energy transfer (EET). (i) For certain fold numbers, there exist optimal basis cells with rotational symmetry, extendable to the entire lattice for the global optimization of the EET network. (ii) The type of basis cell can reduce or remove the frustration of EET rates across the photosynthetic network. We find that the existence of a basis cell and its type are directly related to the number of matching points S between the fold symmetry and the hexagonal lattice. The two complementary mechanisms provide selection criteria for the fold number and identify groups of consecutive numbers. Remarkably, one such group consists of the naturally occurring 8-, 9-, and 10-fold rings. By considering the inter-ring distance and EET rate, we demonstrate that this group can achieve minimal rotational sensitivity in addition to an optimal packing density, achieving robust and efficient EET. This corroborates our findings i and ii and, through their direct relation to S, suggests the design principle of matching the internal symmetry with the lattice order. PMID:23650366

  3. Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae

    PubMed Central

    2013-01-01

    Background Xanthophylls, oxygenated derivatives of carotenes, play critical roles in photosynthetic apparatus of cyanobacteria, algae, and higher plants. Although the xanthophylls biosynthetic pathway of algae is largely unknown, it is of particular interest because they have a very complicated evolutionary history. Carotenoid hydroxylase (CHY) is an important protein that plays essential roles in xanthophylls biosynthesis. With the availability of 18 sequenced algal genomes, we performed a comprehensive comparative analysis of chy genes and explored their distribution, structure, evolution, origins, and expression. Results Overall 60 putative chy genes were identified and classified into two major subfamilies (bch and cyp97) according to their domain structures. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. The homologs of cyp97a/c genes were widespread only in green algae, while cyp97b paralogs were seen in most of algae. Phylogenetic analysis on cyp97 genes supported the hypothesis that cyp97b is an ancient gene originated before the formation of extant algal groups. The cyp97a gene is more closely related to cyp97c in evolution than to cyp97b. The two cyp97 genes were isolated from the green alga Haematococcus pluvialis, and transcriptional expression profiles of chy genes were observed under high light stress of different wavelength. Conclusions Green algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. The origination of cyp97a/c is most likely due to gene duplication before divergence of

  4. Photosynthetic Gene Expression in Meristems and during Initial Leaf Development in a C4 Dicotyledonous Plant.

    PubMed

    Ramsperger, V. C.; Summers, R. G.; Berry, J. O.

    1996-08-01

    Immunolocalization and fluorescent in situ hybridization were used with confocal microscopy to examine patterns of photosynthetic gene expression during initial stages of leaf development in the C4 dicot Amaranthus hypochondriacus. mRNAs encoding the large and small subunit of ribulose-1,5-bisphosphate carboxylase were present in the apical dome and in all cells of the leaf primordia. In contrast, these polypeptides were detected only in cells of the ground meristem, with no accumulation detected in the apical dome or in other leaf primordia cells. The ribulose-1,5-bisphosphate carboxylase transcripts showed very little cell-type specificity as leaf structures began to differentiate, whereas their polypeptides accumulated primarily in bundle-sheath precursor cells. Phosphoenolpyruvate carboxylase and pyruvate orthophosphate dikinase mRNAs were abundant in meristems and leaf primordia, although their corresponding polypeptides did not accumulate in leaves until the leaf vascular system began to differentiate. These polypeptides were mostly restricted to premesophyll cells from their earliest detection, whereas their transcripts remained present in nearly all leaf cells. These findings indicate that individual C4 genes are independently regulated as they become initially localized to their appropriate cell types. Furthermore, posttranscriptional regulation plays a major role in determining early patterns of C4 gene expression. PMID:12226343

  5. An Evaluation of Sensor Performance for Harmful Compounds by Using Photo-Induced Electron Transfer from Photosynthetic Membranes to Electrodes.

    PubMed

    Kasuno, Megumi; Kimura, Hiroki; Yasutomo, Hisataka; Torimura, Masaki; Murakami, Daisuke; Tsukatani, Yusuke; Hanada, Satoshi; Matsushita, Takayuki; Tao, Hiroaki

    2016-01-01

    Rapid, simple, and low-cost screening procedures are necessary for the detection of harmful compounds in the effluent that flows out of point sources such as industrial outfall. The present study investigated the effects on a novel sensor of harmful compounds such as KCN, phenol, and herbicides such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), and 2-N-tert-butyl-4-N-ethyl-6-methylsulfanyl-1,3,5-triazine-2,4-diamine (terbutryn). The sensor employed an electrode system that incorporated the photocurrent of intra-cytoplasmic membranes (so-called chromatophores) prepared from photosynthetic bacteria and linked using carbon paste electrodes. The amperometric curve (photocurrent-time curve) of photo-induced electron transfer from chromatophores of the purple photosynthetic bacterium Rhodobacter sphaeroides to the electrode via an exogenous electron acceptor was composed of two characteristic phases: an abrupt increase in current immediately after illumination (I₀), and constant current over time (Ic). Compared with other redox compounds, 2,5-dichloro-1,4-benzoquinone (DCBQ) was the most useful exogenous electron acceptor in this system. Photo-reduction of DCBQ exhibited Michaelis-Menten-like kinetics, and reduction rates were dependent on the amount of DCBQ and the photon flux intensity. The Ic decreased in the presence of KCN at concentrations over 0.05 μM (=μmol·dm(-3)). The I₀ decreased following the addition of phenol at concentrations over 20 μM. The Ic was affected by terbutryn at concentrations over 10 μM. In contrast, DCMU and atrazine had no effect on either I₀ or Ic. The utility of this electrode system for the detection of harmful compounds is discussed. PMID:27023553

  6. An Evaluation of Sensor Performance for Harmful Compounds by Using Photo-Induced Electron Transfer from Photosynthetic Membranes to Electrodes

    PubMed Central

    Kasuno, Megumi; Kimura, Hiroki; Yasutomo, Hisataka; Torimura, Masaki; Murakami, Daisuke; Tsukatani, Yusuke; Hanada, Satoshi; Matsushita, Takayuki; Tao, Hiroaki

    2016-01-01

    Rapid, simple, and low-cost screening procedures are necessary for the detection of harmful compounds in the effluent that flows out of point sources such as industrial outfall. The present study investigated the effects on a novel sensor of harmful compounds such as KCN, phenol, and herbicides such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), and 2-N-tert-butyl-4-N-ethyl-6-methylsulfanyl-1,3,5-triazine-2,4-diamine (terbutryn). The sensor employed an electrode system that incorporated the photocurrent of intra-cytoplasmic membranes (so-called chromatophores) prepared from photosynthetic bacteria and linked using carbon paste electrodes. The amperometric curve (photocurrent-time curve) of photo-induced electron transfer from chromatophores of the purple photosynthetic bacterium Rhodobacter sphaeroides to the electrode via an exogenous electron acceptor was composed of two characteristic phases: an abrupt increase in current immediately after illumination (I0), and constant current over time (Ic). Compared with other redox compounds, 2,5-dichloro-1,4-benzoquinone (DCBQ) was the most useful exogenous electron acceptor in this system. Photo-reduction of DCBQ exhibited Michaelis-Menten-like kinetics, and reduction rates were dependent on the amount of DCBQ and the photon flux intensity. The Ic decreased in the presence of KCN at concentrations over 0.05 μM (=μmol·dm−3). The I0 decreased following the addition of phenol at concentrations over 20 μM. The Ic was affected by terbutryn at concentrations over 10 μM. In contrast, DCMU and atrazine had no effect on either I0 or Ic. The utility of this electrode system for the detection of harmful compounds is discussed. PMID:27023553

  7. Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures

    PubMed Central

    Pinhassi, Roy I.; Larom, Shirley; Linkov, Artyom; Boulouis, Alix; Schöttler, Mark-Aurel; Bock, Ralph; Rothschild, Avner; Adir, Noam; Schuster, Gadi

    2015-01-01

    Thylakoid membranes contain the redox active complexes catalyzing the light-dependent reactions of photosynthesis in cyanobacteria, algae and plants. Crude thylakoid membranes or purified photosystems from different organisms have previously been utilized for generation of electrical power and/or fuels. Here we investigate the electron transferability from thylakoid preparations from plants or the cyanobacterium Synechocystis. We show that upon illumination, crude Synechocystis thylakoids can reduce cytochrome c. In addition, this crude preparation can transfer electrons to a graphite electrode, producing an unmediated photocurrent of 15 μA/cm2. Photocurrent could be obtained in the presence of the PSII inhibitor DCMU, indicating that the source of electrons is QA, the primary Photosystem II acceptor. In contrast, thylakoids purified from plants could not reduce cyt c, nor produced a photocurrent in the photocell in the presence of DCMU. The production of significant photocurrent (100 μA/cm2) from plant thylakoids required the addition of the soluble electron mediator DCBQ. Furthermore, we demonstrate that use of crude thylakoids from the D1-K238E mutant in Synechocystis resulted in improved electron transferability, increasing the direct photocurrent to 35 μA/cm2. Applying the analogous mutation to tobacco plants did not achieve an equivalent effect. While electron abstraction from crude thylakoids of cyanobacteria or plants is feasible, we conclude that the site of the abstraction of the electrons from the thylakoids, the architecture of the thylakoid preparations influence the site of the electron abstraction, as well as the transfer pathway to the electrode. This dictates the use of different strategies for production of sustainable electrical current from photosynthetic thylakoid membranes of cyanobacteria or higher plants. PMID:25915422

  8. Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures.

    PubMed

    Pinhassi, Roy I; Kallmann, Dan; Saper, Gadiel; Larom, Shirley; Linkov, Artyom; Boulouis, Alix; Schöttler, Mark-Aurel; Bock, Ralph; Rothschild, Avner; Adir, Noam; Schuster, Gadi

    2015-01-01

    Thylakoid membranes contain the redox active complexes catalyzing the light-dependent reactions of photosynthesis in cyanobacteria, algae and plants. Crude thylakoid membranes or purified photosystems from different organisms have previously been utilized for generation of electrical power and/or fuels. Here we investigate the electron transferability from thylakoid preparations from plants or the cyanobacterium Synechocystis. We show that upon illumination, crude Synechocystis thylakoids can reduce cytochrome c. In addition, this crude preparation can transfer electrons to a graphite electrode, producing an unmediated photocurrent of 15 μA/cm2. Photocurrent could be obtained in the presence of the PSII inhibitor DCMU, indicating that the source of electrons is QA, the primary Photosystem II acceptor. In contrast, thylakoids purified from plants could not reduce cyt c, nor produced a photocurrent in the photocell in the presence of DCMU. The production of significant photocurrent (100 μA/cm2) from plant thylakoids required the addition of the soluble electron mediator DCBQ. Furthermore, we demonstrate that use of crude thylakoids from the D1-K238E mutant in Synechocystis resulted in improved electron transferability, increasing the direct photocurrent to 35 μA/cm2. Applying the analogous mutation to tobacco plants did not achieve an equivalent effect. While electron abstraction from crude thylakoids of cyanobacteria or plants is feasible, we conclude that the site of the abstraction of the electrons from the thylakoids, the architecture of the thylakoid preparations influence the site of the electron abstraction, as well as the transfer pathway to the electrode. This dictates the use of different strategies for production of sustainable electrical current from photosynthetic thylakoid membranes of cyanobacteria or higher plants. PMID:25915422

  9. Membrane Sequestration of PII Proteins and Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus▿

    PubMed Central

    Tremblay, Pier-Luc; Drepper, Thomas; Masepohl, Bernd; Hallenbeck, Patrick C.

    2007-01-01

    Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the R. capsulatus membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a glnK mutant, more GlnB was found to be membrane associated under these conditions. In a glnB mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in R. capsulatus. PMID:17586647

  10. Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

    PubMed Central

    Cartron, Michaël L.; Olsen, John D.; Sener, Melih; Jackson, Philip J.; Brindley, Amanda A.; Qian, Pu; Dickman, Mark J.; Leggett, Graham J.; Schulten, Klaus; Hunter, C. Neil

    2014-01-01

    Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities. PMID:24530865

  11. Analysis of the Expression and Activity of Nitric Oxide Synthase from Marine Photosynthetic Microorganisms.

    PubMed

    Foresi, Noelia; Correa-Aragunde, Natalia; Santolini, Jerome; Lamattina, Lorenzo

    2016-01-01

    Nitric oxide (NO) functions as a signaling molecule in many biological processes in species belonging to all kingdoms of life. In animal cells, NO is synthesized primarily by NO synthase (NOS), an enzyme that catalyze the NADPH-dependent oxidation of L-arginine to NO and L-citrulline. Three NOS isoforms have been identified, the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) and one inducible (iNOS). Plant NO synthesis is complex and is a matter of ongoing investigation and debate. Despite evidence of an Arg-dependent pathway for NO synthesis in plants, no plant NOS homologs to animal forms have been identified to date. In plants, there is also evidence for a nitrate-dependent mechanism of NO synthesis, catalyzed by cytosolic nitrate reductase. The existence of a NOS enzyme in the plant kingdom, from the tiny single-celled green alga Ostreococcus tauri was reported in 2010. O. tauri shares a common ancestor with higher plants and is considered to be part of an early diverging class within the green plant lineage.In this chapter we describe detailed protocols to study the expression and characterization of the enzymatic activity of NOS from O. tauri. The most used methods for the characterization of a canonical NOS are the analysis of spectral properties of the oxyferrous complex in the heme domain, the oxyhemoglobin (oxyHb) and citrulline assays and the NADPH oxidation for in vitro analysis of its activity or the use of fluorescent probes and Griess assay for in vivo NO determination. We further discuss the advantages and drawbacks of each method. Finally, we remark factors associated to the measurement of NOS activity in photosynthetic organisms that can generate misunderstandings in the interpretation of results. PMID:27094418

  12. The design and synthesis of artificial photosynthetic antennas, reaction centres and membranes.

    PubMed Central

    Moore, T A; Moore, A L; Gust, D

    2002-01-01

    Artificial antenna systems and reaction centres synthesized in our laboratory are used to illustrate that structural and thermodynamic factors controlling energy and electron transfer in these constructs can be modified to optimize performance. Artificial reaction centres have been incorporated into liposomal membranes where they convert light energy to vectorial redox potential. This redox potential drives a Mitchellian, quinone-based, proton-transporting redox loop that generates a Deltamu H(+) of ca. 4.4 kcal mol(-1) comprising DeltapH ca. 2.1 and Deltapsi ca. 70 mV. In liposomes containing CF(0)F(1)-ATP synthase, this system drives ATP synthesis against an ATP chemical potential similar to that observed in natural systems. PMID:12437888

  13. Synchrotron Small-Angle X-Ray Scattering Investigation on Integral Membrane Protein Light-Harvesting Complex LH2 from Photosynthetic Bacterium Rhodopseudomonas Acidophila

    NASA Astrophysics Data System (ADS)

    Du, Lu-Chao; Weng, Yu-Xiang; Hong, Xin-Guo; Xian, Ding-Chang; Kobayashi, Katsumi

    2006-07-01

    Structures of membrane protein in solution are different from that in crystal phase. We present the primary results of small angle x-ray scattering (SAXS) resolved topological structures of a light harvesting antenna membrane protein complex LH2 from photosynthetic bacteria Rhodopseudomonas acidophila in detergent solution for the first time. Our results show that the elliptical shape of the LH2 complex in solution clearly deviates from its circular structure in crystal phase determined by x-ray diffraction. This result provides an insight into the structure and function interplay in LH2.

  14. cor Gene Expression in Barley Mutants Affected in Chloroplast Development and Photosynthetic Electron Transport1

    PubMed Central

    Dal Bosco, Cristina; Busconi, Marco; Govoni, Chiara; Baldi, Paolo; Stanca, A. Michele; Crosatti, Cristina; Bassi, Roberto; Cattivelli, Luigi

    2003-01-01

    The expression of several barley (Hordeum vulgare) cold-regulated (cor) genes during cold acclimation was blocked in the albino mutant an, implying a chloroplast control on mRNAs accumulation. By using albino and xantha mutants ordered according to the step in chloroplast biogenesis affected, we show that the cold-dependent accumulation of cor14b, tmc-ap3, and blt14 mRNAs depends on plastid developmental stage. Plants acquire the ability to fully express cor genes only after the development of primary thylakoid membranes in their chloroplasts. To investigate the chloroplast-dependent mechanism involved in cor gene expression, the activity of a 643-bp cor14b promoter fragment was assayed in wild-type and albino mutant an leaf explants using transient β-glucuronidase reporter expression assay. Deletion analysis identified a 27-bp region between nucleotides −274 and −247 with respect to the transcription start point, encompassing a boundary of some element that contributes to the cold-induced expression of cor14b. However, cor14b promoter was equally active in green and in albino an leaves, suggesting that chloroplast controls cor14b expression by posttranscriptional mechanisms. Barley mutants lacking either photosystem I or II reaction center complexes were then used to evaluate the effects of redox state of electron transport chain components on COR14b accumulation. In the mutants analyzed, the amount of COR14b protein, but not the steady-state level of the corresponding mRNA, was dependent on the redox state of the electron transport chain. Treatments of the vir-zb63 mutant with electron transport chain inhibitors showed that oxidized plastoquinone promotes COR14b accumulation, thus suggesting a molecular relationship between plastoquinone/plastoquinol pool and COR14b. PMID:12586903

  15. Effect of partial or complete elimination of light-harvesting complexes on the surface electric properties and the functions of cyanobacterial photosynthetic membranes.

    PubMed

    Dobrikova, Anelia G; Domonkos, Ildikó; Sözer, Özge; Laczkó-Dobos, Hajnalka; Kis, Mihály; Párducz, Árpád; Gombos, Zoltán; Apostolova, Emilia L

    2013-02-01

    Influence of the modification of the cyanobacterial light-harvesting complex [i.e. phycobilisomes (PBS)] on the surface electric properties and the functions of photosynthetic membranes was investigated. We used four PBS mutant strains of Synechocystis sp. PCC6803 as follows: PAL (PBS-less), CK (phycocyanin-less), BE (PSII-PBS-less) and PSI-less/apcE(-) (PSI-less with detached PBS). Modifications of the PBS content lead to changes in the cell morphology and surface electric properties of the thylakoid membranes as well as in their functions, such as photosynthetic oxygen-evolving activity, P700 kinetics and energy transfer between the pigment-protein complexes. Data reveal that the complete elimination of PBS in the PAL mutant causes a slight decrease in the electric dipole moments of the thylakoid membranes, whereas significant perturbations of the surface charges were registered in the membranes without assembled PBS-PSII macrocomplex (BE mutant) or PSI complex (PSI-less mutant). These observations correlate with the detected alterations in the membrane structural organization. Using a polarographic oxygen rate electrode, we showed that the ratio of the fast to the slow oxygen-evolving PSII centers depends on the partial or complete elimination of light-harvesting complexes, as the slow operating PSII centers dominate in the PBS-less mutant and in the mutant with detached PBS. PMID:22582961

  16. Expression strategies for structural studies of eukaryotic membrane proteins.

    PubMed

    Lyons, Joseph A; Shahsavar, Azadeh; Paulsen, Peter Aasted; Pedersen, Bjørn Panyella; Nissen, Poul

    2016-06-01

    Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy. PMID:27362979

  17. Tracking energy transfer between light harvesting complex 2 and 1 in photosynthetic membranes grown under high and low illumination

    PubMed Central

    Lüer, Larry; Moulisová, Vladimíra; Henry, Sarah; Polli, Dario; Brotosudarmo, Tatas H. P.; Hoseinkhani, Sajjad; Brida, Daniele; Lanzani, Guglielmo; Cerullo, Giulio; Cogdell, Richard J.

    2012-01-01

    Energy transfer (ET) between B850 and B875 molecules in light harvesting complexes LH2 and LH1/RC (reaction center) complexes has been investigated in membranes of Rhodopseudomonas palustris grown under high- and low-light conditions. In these bacteria, illumination intensity during growth strongly affects the type of LH2 complexes synthesized, their optical spectra, and their amount of energetic disorder. We used a specially built femtosecond spectrometer, combining tunable narrowband pump with broadband white-light probe pulses, together with an analytical method based on derivative spectroscopy for disentangling the congested transient absorption spectra of LH1 and LH2 complexes. This procedure allows real-time tracking of the forward (LH2 → LH1) and backward (LH2←LH1) ET processes and unambiguous determination of the corresponding rate constants. In low-light grown samples, we measured lower ET rates in both directions with respect to high-light ones, which is explained by reduced spectral overlap between B850 and B875 due to partial redistribution of oscillator strength into a higher energetic exciton transition. We find that the low-light adaptation in R. palustris leads to a reduced elementary backward ET rate, in accordance with the low probability of two simultaneous excitations reaching the same LH1/RC complex under weak illumination. Our study suggests that backward ET is not just an inevitable consequence of vectorial ET with small energetic offsets, but is in fact actively managed by photosynthetic bacteria. PMID:22307601

  18. Tracking energy transfer between light harvesting complex 2 and 1 in photosynthetic membranes grown under high and low illumination.

    PubMed

    Lüer, Larry; Moulisová, Vladimíra; Henry, Sarah; Polli, Dario; Brotosudarmo, Tatas H P; Hoseinkhani, Sajjad; Brida, Daniele; Lanzani, Guglielmo; Cerullo, Giulio; Cogdell, Richard J

    2012-01-31

    Energy transfer (ET) between B850 and B875 molecules in light harvesting complexes LH2 and LH1/RC (reaction center) complexes has been investigated in membranes of Rhodopseudomonas palustris grown under high- and low-light conditions. In these bacteria, illumination intensity during growth strongly affects the type of LH2 complexes synthesized, their optical spectra, and their amount of energetic disorder. We used a specially built femtosecond spectrometer, combining tunable narrowband pump with broadband white-light probe pulses, together with an analytical method based on derivative spectroscopy for disentangling the congested transient absorption spectra of LH1 and LH2 complexes. This procedure allows real-time tracking of the forward (LH2 → LH1) and backward (LH2←LH1) ET processes and unambiguous determination of the corresponding rate constants. In low-light grown samples, we measured lower ET rates in both directions with respect to high-light ones, which is explained by reduced spectral overlap between B850 and B875 due to partial redistribution of oscillator strength into a higher energetic exciton transition. We find that the low-light adaptation in R. palustris leads to a reduced elementary backward ET rate, in accordance with the low probability of two simultaneous excitations reaching the same LH1/RC complex under weak illumination. Our study suggests that backward ET is not just an inevitable consequence of vectorial ET with small energetic offsets, but is in fact actively managed by photosynthetic bacteria. PMID:22307601

  19. Phylogeny of Galactolipid Synthase Homologs Together with their Enzymatic Analyses Revealed a Possible Origin and Divergence Time for Photosynthetic Membrane Biogenesis

    PubMed Central

    Yuzawa, Yuichi; Nishihara, Hidenori; Haraguchi, Tsuyoshi; Masuda, Shinji; Shimojima, Mie; Shimoyama, Atsushi; Yuasa, Hideya; Okada, Norihiro; Ohta, Hiroyuki

    2012-01-01

    The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged ∼323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution. PMID:22210603

  20. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

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

    SciTech Connect

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

    2009-01-01

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

  2. Clade-Specific Quantitative Analysis of Photosynthetic Gene Expression in Prochlorococcus

    PubMed Central

    Fernández-Pinos, María-Carmen; Casado, Marta; Caballero, Gemma; Zinser, Erik R.; Dachs, Jordi; Piña, Benjamin

    2015-01-01

    Newly designed primers targeting rbcL (CO2 fixation), psbA (photosystem II) and rnpB (reference) genes were used in qRT-PCR assays to assess the photosynthetic capability of natural communities of Prochlorococcus, the most abundant photosynthetic organism on Earth and a major contributor to primary production in oligotrophic oceans. After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths. Sequence and quantitative analyses of the corresponding amplicons showed the presence of high-light (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences. Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons. Laboratory exposure of Prochlorococcus MED4 (HL) and MIT9313 (LL) strains to organic pollutants (PAHs and organochlorine compounds) showed a decrease of rbcL transcript abundances, and of the rbcL to psbA ratios for both strains. We propose this technique as a convenient assay to evaluate effects of environmental stressors, including pollution, on the oceanic Prochlorococcus photosynthetic function. PMID:26244890

  3. Clade-Specific Quantitative Analysis of Photosynthetic Gene Expression in Prochlorococcus.

    PubMed

    Fernández-Pinos, María-Carmen; Casado, Marta; Caballero, Gemma; Zinser, Erik R; Dachs, Jordi; Piña, Benjamin

    2015-01-01

    Newly designed primers targeting rbcL (CO2 fixation), psbA (photosystem II) and rnpB (reference) genes were used in qRT-PCR assays to assess the photosynthetic capability of natural communities of Prochlorococcus, the most abundant photosynthetic organism on Earth and a major contributor to primary production in oligotrophic oceans. After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths. Sequence and quantitative analyses of the corresponding amplicons showed the presence of high-light (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences. Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons. Laboratory exposure of Prochlorococcus MED4 (HL) and MIT9313 (LL) strains to organic pollutants (PAHs and organochlorine compounds) showed a decrease of rbcL transcript abundances, and of the rbcL to psbA ratios for both strains. We propose this technique as a convenient assay to evaluate effects of environmental stressors, including pollution, on the oceanic Prochlorococcus photosynthetic function. PMID:26244890

  4. Short-term UV-B radiation affects photosynthetic performance and antioxidant gene expression in highbush blueberry leaves.

    PubMed

    Inostroza-Blancheteau, Claudio; Acevedo, Patricio; Loyola, Rodrigo; Arce-Johnson, Patricio; Alberdi, Miren; Reyes-Díaz, Marjorie

    2016-10-01

    The impact of increased artificial UV-B radiation on photosynthetic performance, antioxidant and SOD activities and molecular antioxidant metabolism responses in leaves of two highbush blueberry (Vaccinium corymbosum L. cv. Brigitta and Bluegold) genotypes was studied. Plants were grown in a solid substrate and exposed to 0, 0.07, 0.12 and 0.19 W m(-2) of biologically-effective UV-B irradiance for 0-72 h. Our findings show that net photosynthesis (Pn) decreased significantly in Bluegold, accompanied by a reduction in the effective quantum yield (ФPSII) and electron transport rate (ETR), especially at the highest UV-B irradiation. On the other hand, Brigitta showed a better photosynthetic performance, as well as a clear increment in the antioxidant activity response that could be associated with increased superoxide dismutase activity (SOD) in the early hours of induced UV-B stress in all treatments. At the molecular level, the expression of the three antioxidant genes evaluated in both genotypes had a similar tendency. However, ascorbate peroxidase (APX) expression was significantly increased (6-fold) in Bluegold compared to Brigitta. Thus, the reduction of Pn concomitant with a lower photochemical performance and a reduced response of antioxidant metabolism suggest that the Bluegold genotype is more sensitive to UV-B radiation, while Brigitta appears to tolerate better moderate UV-B irradiance in a short-term experiment. PMID:27343876

  5. Translation Levels Control Multi-Spanning Membrane Protein Expression

    PubMed Central

    Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

    2012-01-01

    Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. PMID:22563408

  6. Effects of exogenous spermidine on photosynthetic capacity and expression of Calvin cycle genes in salt-stressed cucumber seedlings.

    PubMed

    Shu, Sheng; Chen, Lifang; Lu, Wei; Sun, Jin; Guo, Shirong; Yuan, Yinhui; Li, Jun

    2014-11-01

    We investigated the effects of exogenous spermidine (Spd) on growth, photosynthesis and expression of the Calvin cycle-related genes in cucumber seedlings (Cucumis sativus L.) exposed to NaCl stress. Salt stress reduced net photosynthetic rates (PN), actual photochemical efficiency of PSII (ΦPSII) and inhibited plant growth. Application of exogenous Spd to salinized nutrient solution alleviated salinity-induced the inhibition of plant growth, together with an increase in PN and ΦPSII. Salinity markedly reduced the maximum carboxylase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Vcmax), the maximal velocity of RuBP regeneration (Jmax), triose-phosphate utilization capacity (TPU) and carboxylation efficiency (CE). Spd alleviated the negative effects on CO2 assimilation induced by salt stress. Moreover, Spd significantly increased the activities and contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and fructose-1,6-biphosphate aldolase (ALD; aldolase) in the salt-stressed cucumber leaves. On the other hand, salinity up-regulated the transcriptional levels of ribulose-1,5-bisphosphate (RCA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribrokinase (PRK) and down-regulated the transcriptional levels of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RbcL), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS), ALD, triose-3-phosphate isomerase (TPI), fructose-1,6-bisphosphate phosphatase (FBPase) and 3-phosphoglyceric acid kinase (PGK). However, Spd application to salt-stressed plant roots counteracted salinity-induced mRNA expression changes in most of the above-mentioned genes. These results suggest that Spd could improve photosynthetic capacity through regulating gene expression and activity of key enzymes for CO2 fixation, thus confers tolerance to salinity on cucumber plants. PMID:25069716

  7. Giant vesicles functionally expressing membrane receptors for an insect pheromone.

    PubMed

    Hamada, Satoshi; Tabuchi, Masashi; Toyota, Taro; Sakurai, Takeshi; Hosoi, Tomohiro; Nomoto, Tomonori; Nakatani, Kei; Fujinami, Masanori; Kanzaki, Ryohei

    2014-03-18

    To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor-surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone. PMID:24509495

  8. Expression of natural antimicrobials by human placenta and fetal membranes.

    PubMed

    King, A E; Paltoo, A; Kelly, R W; Sallenave, J-M; Bocking, A D; Challis, J R G

    2007-01-01

    Preterm birth associated with infection is a major clinical problem. We hypothesized that this condition is associated with altered expression of natural antimicrobial molecules (beta-defensins (HBD), elafin). Therefore, we examined expression of these molecules and their regulation by proinflammatory cytokines in placentae and fetal membranes from term pregnancy. HBD1-3 and elafin were localized by immunohistochemistry in fetal membranes and placenta. Real-time quantitative PCR was used to examine mRNA expression in primary trophoblast cells treated with inflammatory molecules. HBD1-3 and elafin were immunolocalized to placental and chorion trophoblast layers of fetal membranes and placenta. Immunoreactivity was also observed in amnion epithelium and decidua. No differences were noted between samples from women who were not in labour compared to those in active labour. In in vitro cultures of primary trophoblast cells, HBD2 and elafin mRNA expression was upregulated by the proinflammatory cytokine, IL-1beta. These results suggest that the chorion and placental trophoblast layers may be key barriers to the progression of infection in the pregnant uterus. Natural antimicrobial expression may be altered in response to inflammatory mediator expression associated with the onset of labour and/or uterine infection, providing increased protection when the uterus may be particularly susceptible to infection. PMID:16513165

  9. Membrane channel gene expression in human costal and articular chondrocytes

    PubMed Central

    Asmar, A.; Barrett-Jolley, R.; Werner, A.; Kelly, R.; Stacey, M.

    2016-01-01

    ABSTRACT Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  10. Expression of major photosynthetic and salt-resistance genes in invasive reed lineages grown under elevated CO2 and temperature

    PubMed Central

    Eller, Franziska; Lambertini, Carla; Nielsen, Mette W; Radutoiu, Simona; Brix, Hans

    2014-01-01

    It is important to investigate the molecular causes of the variation in ecologically important traits to fully understand phenotypic responses to climate change. In the Mississippi River Delta, two distinct, sympatric invasive lineages of common reed (Phragmites australis) are known to differ in several ecophysiological characteristics and are expected to become more salt resistant due to increasing atmospheric CO2 and temperature. We investigated whether different patterns of gene expression can explain their ecophysiological differences and increased vigor under future climatic conditions. We compared the transcript abundance of photosynthetic genes of the Calvin cycle (Rubisco small subunit, RbcS; Phosphoglycerate kinase, PGK; Phosphoribulokinase, PRK), genes related with salt transport (Na+/H+ antiporter, PhaNHA) and oxidative stress response genes (Manganese Superoxide dismutase, MnSOD; Glutathione peroxidase, GPX), and the total aboveground biomass production between two genotypes representing the two lineages. The two genotypes (Delta-type, Mediterranean lineage, and EU-type, Eurasian lineage) were grown under an ambient and a future climate scenario with simultaneously elevated CO2 and temperature, and under two different soil salinities (0‰ or 20‰). We found neither differences in the aboveground biomass production nor the transcript abundances of the two genotypes, but soil salinity significantly affected all the investigated parameters, often interacting with the climatic conditions. At 20‰ salinity, most genes were higher expressed in the future than in the ambient climatic conditions. Higher transcription of the genes suggests higher abundance of the protein they code for, and consequently increased photosynthate production, improved stress responses, and salt exclusion. Therefore, the higher expression of these genes most likely contributed to the significantly ameliorated salinity impact on the aboveground biomass production of both P

  11. Expressing and purifying membrane transport proteins in high yield.

    PubMed

    Hale, Calvin C; Hill, Chananada K; Price, Elmer M; Bossuyt, Julie

    2002-01-01

    Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic. PMID:11741710

  12. Tracing of backward energy transfer from LH1 to LH2 in photosynthetic membranes grown under high and low irradiation.

    NASA Astrophysics Data System (ADS)

    Lüer, L.; Moulisová, V.; Henry, S.; Polli, D.; Brotosudarmo, T. H. P.; Hoseinkhani, S.; Brida, D.; Lanzani, G.; Cerullo, G.; Cogdell, R. J.

    2013-03-01

    By introducing derivative transient absorption spectroscopy, we obtain rate constants for backward and forward energy transfer between LH1 and LH2 complexes in purple bacterial membranes. We find that backward energy transfer is strongly reduced in membranes grown under low irradiation conditions, compared to high light grown ones. We conclude that backward energy transfer is managed actively by the bacteria to avoid LH1 exciton deactivation under high irradiation conditions. The analytical method is generally applicable to excitonically coupled systems.

  13. Expression of Eukaryotic Membrane Proteins in Pichia pastoris.

    PubMed

    Hartmann, Lucie; Kugler, Valérie; Wagner, Renaud

    2016-01-01

    A key point when it comes to heterologous expression of eukaryotic membrane proteins (EMPs) is the choice of the best-suited expression platform. The yeast Pichia pastoris has proven to be a very versatile system showing promising results in a growing number of cases. Indeed, its particular methylotrophic characteristics combined to the very simple handling of a eukaryotic microorganism that possesses the majority of mammalian-like machineries make it a very competitive expression system for various complex proteins, in amounts compatible with functional and structural studies. This chapter describes a set of robust methodologies routinely used for the successful expression of a variety of EMPs, going from yeast transformation with the recombinant plasmid to the analysis of the quality and quantity of the proteins produced. PMID:27485335

  14. Transcriptional Control of Expression of Genes for Photosynthetic Reaction Center and Light-Harvesting Proteins in the Purple Bacterium Rhodovulum sulfidophilum

    PubMed Central

    Masuda, Shinji; Nagashima, Kenji V. P.; Shimada, Keizo; Matsuura, Katsumi

    2000-01-01

    The purple photosynthetic bacterium Rhodovulum sulfidophilum synthesizes photosynthetic apparatus even under highly aerated conditions in the dark. To understand the oxygen-independent expression of photosynthetic genes, the expression of the puf operon coding for the light-harvesting 1 and reaction center proteins was analyzed. Northern blot hybridization analysis showed that puf mRNA synthesis was not significantly repressed by oxygen in this bacterium. High-resolution 5′ mapping of the puf mRNA transcriptional initiation sites and DNA sequence analysis of the puf upstream regulatory region indicated that there are three possible promoters for the puf operon expression, two of which have a high degree of sequence similarity with those of Rhodobacter capsulatus, which shows a high level of oxygen repression of photosystem synthesis. Deletion analysis showed that the third promoter is oxygen independent, but the activity of this promoter was not enough to explain the aerobic level of mRNA. The posttranscriptional puf mRNA degradation is not significantly influenced by oxygen in R. sulfidophilum. From these results, we conclude that puf operon expression in R. sulfidophilum is weakly repressed by oxygen, perhaps as a result of the following: (i) there are three promoters for puf operon transcription, at least one of which is oxygen independent; (ii) readthrough transcripts which may not be affected by oxygen may be significant in maintaining the puf mRNA levels; and (iii) the puf mRNA is fairly stable even under aerobic conditions. PMID:10781546

  15. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  16. Applications of nylon membrane arrays to gene expression analysis.

    PubMed

    Cox, J M

    2001-04-01

    Gene expression analyses by hybridization of probes derived from mRNA to cDNA targets arrayed on a nylon membranes have been performed with increasing frequency and success over the last decade. While the initial costs of generating arrays are moderately high, they are now available commercially as complete packages which include the membranes and associated image analysis software for acquisition and processing of the data. Arrays can be used to generate information concerning the expression of mRNA from cells treated with various agents or from different tissues, e.g. comparing diseased with normal controls. To date, many groups, including immunologists, have used this technology to examine gene expression within their area of biological interest. The main characteristic of these systems is the large amount of data generated, since the expression of many thousands of genes are measured in parallel. The main practical issues are sensitivity of detection, reproducibility, comparability with other systems (e.g. Northern blots) and processing of data. Some of the significant applications of nylon array technology to date are reviewed in this chapter, and with these issues in mind, we include a discussion of our own experiences in this area. PMID:11251218

  17. [Membrane-based photochemical systems as models for photosynthetic cells]. Progress report, February 15, 1990--August 31, 1992

    SciTech Connect

    Hurst, J.K.

    1992-12-31

    The objectives of this research are to improve our conceptual view of the ways in which membranes and interfaces can be used to control chemical reactivity. We have focused on understanding three elementary processes that are central to developing membrane-based integrated chemical systems for water photolysis or related photoconversion/photostorage processes. Specifically, we have sought to identify: the influence of interfaces upon charge separation/recombination reactions, pathways for transmembrane charge separation across hydrocarbon bilayer membranes, and mechanisms of water oxidation catalyzed by transition metal coordination complexes. Historically, the chemical dynamics of each of these processes has been poorly understood, with numerous unresolved issues and conflicting viewpoints appearing in the literature. As described in this report our recent research has led to considerable clarification of the underlying reaction mechanisms.

  18. Membrane-bound c-type cytochromes in Heliobacillus mobilis. In vivo study of the hemes involved in electron donation to the photosynthetic reaction center

    SciTech Connect

    Nitschke, W.; Kramer, D.M.; Liebl, U.

    1995-09-19

    The amount of heme per photosynthetic reaction center (RC) was examined in whole cells of Heliobacillus mobilis, and a stoichiometry of 5-6 hemes c and 1-3 hemes b per RC was found. Virtually the full complement of heme was seen to be functionally connected to the pool of electron donors to the photosynthetic RC. The kinetic parameters of electron transfer between reduced c-type hemes and the photooxidized primary donor P{sub 798}{sup +} were studied in whole cells and membrane fragments. The invivo half-times of electron donation (50% with t{sub 1/2}=110{mu}s, 50% with t{sub 1/2}=600 {mu}s) were seen to slow down to half-times in the range of several and several tens of milliseconds following disruption of cells. A severe conformational lateration or a change in the identity of the donating heme is discussed. Redox titrations of the flash-induced absorption changes performed on whole cells in the presence of mediators yielded the following redox midpoint potentials: P{sub 798}, E{sub m}=+240mV; heme c{sub 553}, E{sub m} = +190, +170, and +90 mV for the heme components oxidized after the first, second, and third flash, respectively. The results demonstrate that the pool of c{sub 553} hemes donating electrons to the RC is heterogeneous and that it consists of either several distinguishable cytochromes or multiheme cytochromes or both. The number of hemes reduced and the kinetics of heme rereduction after flash-induced oxidation were found to depend strongly on the degree of anaerobicity in the interior compartment for the cell. A model rationalizing the obtained results in terms of a set of differing redox components is proposed. 42 refs., 7 figs.

  19. Expression and purification of the membrane enzyme selenoprotein K.

    PubMed

    Liu, Jun; Srinivasan, Prabhavathi; Pham, Diane N; Rozovsky, Sharon

    2012-11-01

    Selenoprotein K (SelK) is a membrane protein residing in the endoplasmic reticulum. The function of SelK is mostly unknown; however, it has been shown to participate in anti-oxidant defense, calcium regulation and in the endoplasmic reticulum associated protein degradation (ERAD) pathway. In order to study the function of SelK and the role of selenocysteine in catalysis, we have tested heterologous expression of human SelK in E. coli. Consequently, we have developed an over-expression strategy that exploits the maltose binding protein as a fusion partner to stabilize and solubilize SelK. The fusion partner can be cleaved from SelK in the presence of a variety of detergents compatible with structural characterization and the protein purified to homogeneity. SelK acquires a helical secondary structure in detergent micelles, even though it was predicted to be an intrinsically disordered protein due to its high percentage of polar residues. The same strategy was successfully applied to preparation of SelK binding partner - selenoprotein S (SelS). Hence, this heterologous expression and purification strategy can be applied to other members of the membrane enzyme family to which SelK belongs. PMID:22963794

  20. Effects of paraquat on photosynthetic pigments, antioxidant enzymes, and gene expression in Chlorella pyrenoidosa under mixotrophic compared with autotrophic conditions.

    PubMed

    Zhang, Weiguo; Liu, Min; Zhang, Peiliang; Yu, Fugen; Lu, Shan; Li, Pengfu; Zhou, Junying

    2014-11-01

    Only limited information is available on herbicide toxicity to algae under mixotrophic conditions. In the present study, we studied the effects of the herbicide paraquat on growth, photosynthetic pigments, antioxidant enzymes, and gene expression in Chlorella pyrenoidosa under mixotrophic compared with autotrophic conditions. The mean measured exposure concentrations of paraquat under mixotrophic and autotrophic conditions were in the range of 0.3-3.4 and 0.6-3.6 μM, respectively. Exposure to paraquat for 72 h under both autotrophic and mixotrophic conditions induced decreased growth and chlorophyll (Chl) content, increased superoxide dismutase and peroxidase activities, and decreased transcript abundances of three photosynthesis-related genes (light-independent protochlorophyllide reductase subunit, photosystem II protein D1, and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit [rbcL]). Compared with autotrophic conditions, the inhibition percentage of growth rate under mixotrophic conditions was lower at 0.8 μM paraquat, whereas it was greater at 1.8 and 3.4 μM paraquat. With exposure to 0.8-3.4 μM paraquat, the inhibition rates of Chl a and b content under mixotrophic conditions (43.1-52.4% and 54.6-59.7%, respectively) were greater compared with autotrophic conditions, whereas the inhibition rate of rbcL gene transcription under mixotrophic conditions (35.7-44.0%) was lower. These data showed that similar to autotrophic conditions, paraquat affected the activities of antioxidant enzymes and decreased Chl synthesis and transcription of photosynthesis-related genes in C. pyrenoidosa under mixotrophic conditions, but a differential susceptibility to paraquat toxicity occurred between autotrophically versus mixotrophically grown cells. PMID:25038722

  1. Expression and purification of integral membrane metallopeptidase HtpX.

    PubMed

    Arolas, Joan L; García-Castellanos, Raquel; Goulas, Theodoros; Akiyama, Yoshinori; Gomis-Rüth, F Xavier

    2014-07-01

    Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-β-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members. PMID:24769134

  2. Bioengineering of photosynthetic membranes. Requirement of magnesium for the conversion of chlorophyllide a to chlorophyll a during the greening of etiochloroplasts in vitro

    SciTech Connect

    Daniell, H.; Rebeiz, C.A.

    1984-01-01

    The massive conversion of delta-aminolevulinic acid (ALA) to protochlorophyllide (Pchlide) and the massive conversion of chlorophyllide a (Chlide a) to chlorophyll a (Chl a) are two essential conditions for the ALA-dependent assembly of photosynthetic membranes in vitro. In this work, the authors describe the development of a cell-free system capable of the forementioned biosynthetic activities at rates higher than in vivo, for the first 2 h of dark-incubation. The cell-free system consisted of 1) etiochloroplasts prepared from kinetin and gibberellic-acid-pretreated cucumber cotyledons, and 2) cofactors and additives described elsewhere and which are needed for the massive conversion of ALA to Pchlide, 3) high concentrations of ATP, MgCl/sub 2/, and an isoprenol alcohol such as phytol, were required for the massive conversion of Chlide a to Chl a. An absolute and novel requirement of Mg/sup 2 +/ for the conversion of Chlide a to Chl a was also demonstrated. In addition to the role of phytol as a substrate for the conversion of Chlide a to Chl a, the data suggested that this alcohol may also be involved in the regulation of the reactions between ALA and Pchlide. It is proposed that during greening, the conversion of Chlide a to Chl a may follow different biosynthetic rates, having different substrate and cofactor requirements, depending on the stage of plastid development.

  3. An integrated study of photochemical function and expression of a key photochemical gene (psbA) in photosynthetic communities of Lake Bonney (McMurdo Dry Valleys, Antarctica).

    PubMed

    Kong, Weidong; Li, Wei; Romancova, Ingrid; Prášil, Ondřej; Morgan-Kiss, Rachael M

    2014-08-01

    Lake Bonney is one of several permanently ice-covered lakes in the McMurdo Dry Valleys, Antarctica, which maintain the only year-round biological activity on the Antarctic continent. Vertically stratified populations of autotrophic microorganisms occupying the water columns are adapted to numerous extreme conditions, including very low light, hypersalinity, ultra-oligotrophy and low temperatures. In this study, we integrated molecular biology, microscopy, flow cytometry, and functional photochemical analyses of the photosynthetic communities residing in the east and west basins of dry valley Lake Bonney. Diversity and abundance of the psbA gene encoding a major protein of the photosystem II reaction center were monitored during the seasonal transition between Antarctic summer (24-h daylight) to winter (24-h darkness). Vertical trends through the photic zone in psbA abundance (DNA and mRNA) closely matched that of primary production in both lobes. Seasonal trends in psbA transcripts differed between the two lobes, with psbA expression in the west basin exhibiting a transient rise in early Fall. Last, using spectroscopic and flow cytometric analyses, we provide the first evidence that the Lake Bonney photosynthetic community is dominated by picophytoplankton that possess photosynthetic apparatus adapted to extreme shade. PMID:24499459

  4. Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat.

    PubMed

    Zhang, HuiFang; Xu, WeiGang; Wang, HuiWei; Hu, Lin; Li, Yan; Qi, XueLi; Zhang, Lei; Li, ChunXin; Hua, Xia

    2014-09-01

    Using particle bombardment transformation, we introduced maize pepc cDNA encoding phosphoenolpyruvate carboxylase (PEPC) and ppdk cDNA encoding pyruvate orthophosphate dikinase (PPDK) into the C3 crop wheat to generate transgenic wheat lines carrying cDNA of pepc (PC lines), ppdk (PK lines) or both (PKC lines). The integration, transcription, and expression of the foreign genes were confirmed by Southern blot, Real-time quantitative reverse transcription PCR (Q-RT-PCR), and Western blot analysis. Q-RT-PCR results indicated that the average relative expression levels of pepc and ppdk in the PKC lines reached 10 and 4.6, respectively, compared to their expressions in untransformed plants (set to 1). The enzyme activities of PEPC and PPDK in the PKC lines were 4.3- and 2.1-fold higher, respectively, than in the untransformed control. The maximum daily net photosynthetic rates of the PKC, PC, and PK lines were enhanced by 26.4, 13.3, and 4.5%, respectively, whereas the diurnal accumulations of photosynthesis were 21.3, 13.9, and 6.9%, respectively, higher than in the control. The Fv/Fm of the transgenic plants decreased less than in the control under high temperature and high light conditions (2 weeks after anthesis), suggesting that the transgenic wheat transports more absorbed light energy into a photochemical reaction. The exogenous maize C4-specific pepc gene was more effective than ppdk at improving the photosynthetic performance and yield characteristics of transgenic wheat, while the two genes showed a synergistic effect when they were transformed into the same genetic background, because the PKC lines exhibited improved photosynthetic and physiological traits. PMID:24595619

  5. Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow.

    PubMed

    Blanco, Nicolás E; Ceccoli, Romina D; Vía, María V Dalla; Voss, Ingo; Segretin, María E; Bravo-Almonacid, Fernando F; Melzer, Michael; Hajirezaei, Mohammad-Reza; Scheibe, Renate; Hanke, Guy T

    2013-02-01

    Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms. PMID:23370717

  6. Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.

    PubMed

    Bird, Louise E; Nettleship, Joanne E; Järvinen, Valtteri; Rada, Heather; Verma, Anil; Owens, Raymond J

    2016-01-01

    The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years. PMID:27553231

  7. Selecting Optimum Eukaryotic Integral Membrane Proteins for Structure Determination by Rapid Expression and Solubilization Screening

    PubMed Central

    Li, Min; Hays, Franklin A.; Roe-Zurz, Zygy; Vuong, Linda; Kelly, Libusha; Ho, Chi-Min; Robbins, Renée M.; Pieper, Ursula; O’Connell, Joseph D.; Miercke, Larry J. W.; Giacomini, Kathleen M.; Sali, Andrej; Stroud, Robert M.

    2009-01-01

    A medium throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from the budding yeast Saccharomyces cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism’s membrane proteome. 272 targets were expressed and of these 234 solubilized in the detergent n-dodecyl-β-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of ten human integral membrane proteins from the solute carrier superfamily (SLC). This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others. PMID:19061901

  8. Analysis of Porphyra Membrane Transporters Demonstrates Gene Transfer among Photosynthetic Eukaryotes and Numerous Sodium-Coupled Transport Systems1[C][W][OA

    PubMed Central

    Chan, Cheong Xin; Zäuner, Simone; Wheeler, Glen; Grossman, Arthur R.; Prochnik, Simon E.; Blouin, Nicolas A.; Zhuang, Yunyun; Benning, Christoph; Berg, Gry Mine; Yarish, Charles; Eriksen, Renée L.; Klein, Anita S.; Lin, Senjie; Levine, Ira; Brawley, Susan H.; Bhattacharya, Debashish

    2012-01-01

    Membrane transporters play a central role in many cellular processes that rely on the movement of ions and organic molecules between the environment and the cell, and between cellular compartments. Transporters have been well characterized in plants and green algae, but little is known about transporters or their evolutionary histories in the red algae. Here we examined 482 expressed sequence tag contigs that encode putative membrane transporters in the economically important red seaweed Porphyra (Bangiophyceae, Rhodophyta). These contigs are part of a comprehensive transcriptome dataset from Porphyra umbilicalis and Porphyra purpurea. Using phylogenomics, we identified 30 trees that support the expected monophyly of red and green algae/plants (i.e. the Plantae hypothesis) and 19 expressed sequence tag contigs that show evidence of endosymbiotic/horizontal gene transfer involving stramenopiles. The majority (77%) of analyzed contigs encode transporters with unresolved phylogenies, demonstrating the difficulty in resolving the evolutionary history of genes. We observed molecular features of many sodium-coupled transport systems in marine algae, and the potential for coregulation of Porphyra transporter genes that are associated with fatty acid biosynthesis and intracellular lipid trafficking. Although both the tissue-specific and subcellular locations of the encoded proteins require further investigation, our study provides red algal gene candidates associated with transport functions and novel insights into the biology and evolution of these transporters. PMID:22337920

  9. Snorkel: an epitope tagging system for measuring the surface expression of membrane proteins.

    PubMed

    Brown, Michael; Stafford, Lewis J; Onisk, Dale; Joaquim, Tony; Tobb, Alhagie; Goldman, Larissa; Fancy, David; Stave, James; Chambers, Ross

    2013-01-01

    Tags are widely used to monitor a protein's expression level, interactions, protein trafficking, and localization. Membrane proteins are often tagged in their extracellular domains to allow discrimination between protein in the plasma membrane from that in internal pools. Multipass membrane proteins offer special challenges for inserting a tag since the extracellular regions are often composed of small loops and thus inserting an epitope tag risks perturbing the structure, function, or location of the membrane protein. We have developed a novel tagging system called snorkel where a transmembrane domain followed by a tag is appended to the cytoplasmic C-terminus of the membrane protein. In this way the tag is displayed extracellularly, but structurally separate from the membrane protein. We have tested the snorkel tag system on a diverse panel of membrane proteins including GPCRs and ion channels and demonstrated that it reliably allows for monitoring of the surface expression. PMID:24023844

  10. [Effects of exogenous GSH on photosynthetic characteristics and expression of key enzyme genes of CO2 assimilation in leaves of tomato seedlings under NaCl stress].

    PubMed

    2014-09-01

    By spraying tomato leaves with reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione synthesis inhibitor (BSO), respectively, the effects of glutathion-mediated redox state on leaf photosynthesis in tomato under NaCl stress were investigated. The results showed that the application of exogenous GSH significantly induced an increase in reducing power level, in- creased the net photosynthetic rate (Pn), stomatal conductance (g(s)), transpiration rate (Tr), as well as the maximum quantum yield of PS II (Fv/Fm), actual photochemical efficiency of PS II (ΦPS II), photochemical quenching coefficient (q(P)) and non-photochemical quenching coefficient (NPQ), and enhanced the Rubisco activity and expression levels of RbcL, RbcS and RCA genes in leaves of tomato seedlings under NaCl stress. These results suggested that GSH alleviated salt-induced oxidative stress by protecting PS II from damage caused by excess energy, and improving the photochemical efficiency of PS II and dark reaction activity of photosynthesis. Although spraying GSSG decreased the level of reducing power and further aggravated the damage and photoinhibition of the leaf photosynthetic apparatus, Pn was not affected in combined stressed (NaCl and GSSG) plants, which might be due to the up-regulation of expression levels of RbcL and RbcS genes. The application of BSO had no significant effects on redox state, CO2 conductivity capacity and PS II photochemical efficiency in tomato leaves under NaCl stress. However, compared to salt singly stressed plants, BSO application increased Pn, likely due to the up-regulation of Rubisco initial activity and RCA and RbcS expression levels. PMID:25757316

  11. Photosynthetic characteristics of an amphibious plant, Eleocharis vivipara: Expression of C4 and C3 modes in contrasting environments

    PubMed Central

    Ueno, Osamu; Samejima, Muneaki; Muto, Shoshi; Miyachi, Shigetoh

    1988-01-01

    Eleocharis vivipara Link, a freshwater amphibious leafless plant belonging to the Cyperaceae can grow in both terrestrial and submersed aquatic conditions. Two forms of E. vivipara obtained from these contrasting environments were examined for the characteristics associated with C4 and C3 photosynthesis. In the terrestrial form (δ 13C values = -13.5 to -15.4‰, where ‰ is parts per thousand), the culms, which are photosynthetic organs, possess a Kranz-type anatomy typical of C4 plants, and well-developed bundle-sheath cells contain numerous large chloroplasts. In the submersed form (δ 13C value = -25.9‰), the culms possess anatomical features characteristic of submersed aquatic plants, and the reduced bundle-sheath cells contain only a few small chloroplasts. 14C pulse-12C chase experiments showed that the terrestrial form and the submersed form fix carbon by way of the C4 pathway, with aspartate (40%) and malate (35%) as the main primary products, and by way of the C3 pathway, with 3-phosphoglyceric acid (53%) and sugar phosphates (14%) as the main primary products, respectively. The terrestrial form showed photosynthetic enzyme activities typical of the NAD-malic enzyme-C4 subtype, whereas the submersed form showed decreased activities of key C4 enzymes and an increased ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) activity. These data suggest that this species can differentiate into the C4 mode under terrestrial conditions and into the C3 mode under submersed conditions. Images PMID:16593980

  12. Cell-free Expression and In Meso Crystallisation of an Integral Membrane Kinase for Structure Determination

    PubMed Central

    Shah, Syed Tasadaque Ali; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank; Caffrey, Martin

    2014-01-01

    Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a 3-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipidic mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28 Å resolution. The quality of cellular and cell-free expressed kinase samples have been evaluated systematically by comparing i) spectroscopic properties, ii) purity and oligomer formation, iii) lipid content and iv) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved. PMID:25012698

  13. Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination.

    PubMed

    Boland, Coilín; Li, Dianfan; Shah, Syed Tasadaque Ali; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank; Caffrey, Martin

    2014-12-01

    Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved. PMID:25012698

  14. Ribulose-1,5-bisphosphate Carboxylase/oxygenase (RubisCO) Gene Expression and Photosynthetic Activity in Nutrient-enriched Mesocosm Experiments

    NASA Astrophysics Data System (ADS)

    Wyman, M.; Davies, J. T.; Weston, K.; Crawford, D. W.; Purdie, D. A.

    1998-02-01

    The temporal variability in carbon dioxide fixation rates and the relative abundance ofrbcLSmRNA (encoding the large subunit of the Calvin cycle enzyme, RubisCO) was determined for nutrient-stimulated populations of marine phytoplankton enclosed in diatom-dominated and coccolithophorid-dominated mesocosms. Both mesocosms were characterized by successive bloom events that were preceded by marked increases in the level of RubisCO gene expression. In general, maxima inrbcLmRNA abundance showed the strongest temporal covariation with peaks in the value of the photosynthetic parameter PBmax, the chlorophyll-specific maximum rate of CO2fixation. Somewhat looser temporal co-variations were observed between peaks in transcript levels and maxima in chlorophyll concentrations or phytoplankton biomass. The specific contribution of the haptophyteEmiliania huxleyito the overall level of gene expression in the diatom-dominated enclosure was investigated using an homologousrbcLgene probe. The results were compared to data obtained at lower hybridization stringency using a generalrbcLprobe originating from the oceanic cyanobacteriumSynechococcusWH8103. The comparative data suggest that, whereas diatoms made a substantial contribution to the mRNA signal during the initial part of the experiment, the contribution ofE. huxleyito the overall level of gene expression increased as the experiment progressed.

  15. A Link Between Integral Membrane Protein Expression and Simulated Integration Efficiency

    PubMed Central

    Müller, Axel; Tiemann, Katrin; Saladi, Shyam M.; Galimidi, Rachel P.; Zhang, Bin; Clemons, William M.; Miller, Thomas F.

    2016-01-01

    Integral membrane proteins (IMP) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels widely vary and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design. PMID:27524616

  16. A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency.

    PubMed

    Marshall, Stephen S; Niesen, Michiel J M; Müller, Axel; Tiemann, Katrin; Saladi, Shyam M; Galimidi, Rachel P; Zhang, Bin; Clemons, William M; Miller, Thomas F

    2016-08-23

    Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design. PMID:27524616

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

    PubMed

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

    2015-01-01

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

  18. Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

    PubMed

    Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

    2015-11-01

    Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber. PMID:26351149

  19. High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

    PubMed Central

    Bruni, Renato

    2014-01-01

    Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

  20. Heat-shock protein expression on the membrane of T cells undergoing apoptosis.

    PubMed Central

    Poccia, F; Piselli, P; Vendetti, S; Bach, S; Amendola, A; Placido, R; Colizzi, V

    1996-01-01

    Heat-shock proteins (hsp) represent a highly conserved family of proteins, normally localized in the cytoplasm and nucleus, whose expression is induced in situations involving cell stress. This paper reports the unusual translocation of hsp to the cell membrane of T cells undergoing apoptosis. We observed that glucocorticosteroid-induced thymocyte death is associated to the surface expression of hsp 60 and hsp 70 in a discrete fraction of apoptotic cells. hsp surface expression is closely related to a thymic subset of immature CD3low/- T cells. The expression of surface hsp 60 appears early after treatment with dexamethasone (3 hr) whereas the membrane expression of hsp 70 follows different kinetics and peaks later. Morphological analysis of the hsp+ apoptotic cells suggest that this subset represents late-stage apoptotic cells at their minimal volume before fragmentation into apoptotic bodies. Membrane expression of hsp is also associated with apoptosis in peripheral blood mononuclear cells from AIDS patients cultured in vitro. Altogether, we show that a discrete fraction of cells undergoing apoptosis expresses membrane hsp 60 and hsp 70, supporting the hypothesis that apoptosis causes a radical alteration in the expression of cell surface molecules. Surface hsp expressed during apoptosis may constitute a novel immune-context able to generate packages of self- and exogenous antigens, originating from degradation of altered cells. Images Figure 1 Figure 3 PMID:8707351

  1. Screening for lipid requirements of membrane proteins by combining cell-free expression with nanodiscs.

    PubMed

    Henrich, Erik; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free (CF) protein expression has emerged as one of the most efficient production platforms for membrane proteins. Central bottlenecks prevalent in conventional cell-based expression systems such as mistargeting, inclusion body formation, degradation as well as product toxicity can be addressed by taking advantage of the reduced complexity of CF expression systems. However, the open accessibility of CF reactions offers the possibility to design customized artificial expression environments by supplying synthetic hydrophobic compounds such as micelles or membranes of defined composition. The open nature of CF systems therefore generally allows systematic screening approaches for the identification of efficient cotranslational solubilization environments of membrane proteins. Synergies exist in particular with the recently developed nanodisc (ND) technology enabling the synthesis of stable and highly soluble particles containing membrane discs of defined composition. Specific types of lipids frequently modulate folding, stability, and activity of integrated membrane proteins. One recently reported example are phospho-MurNAc-pentapeptide (MraY) translocases that catalyze a crucial step in bacterial peptidoglycan biosynthesis making them interesting as future drug targets. Production of functionally active MraY homologues from most human pathogens in conventional cellular production systems was so far not successful due to their obviously strict lipid dependency for functionally folding. We demonstrate that the combination of CF expression with ND technologies is an efficient strategy for the production of folded MraY translocases, and we present a general protocol for the rapid screening of lipid specificities of membrane proteins. PMID:25857790

  2. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    PubMed

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-01

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation. PMID

  3. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

    PubMed Central

    Round, Ekaterina; Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Borshchevskiy, Valentin; Gordeliy, Valentin

    2015-01-01

    Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. PMID:26046789

  4. Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression

    PubMed Central

    2015-01-01

    Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins. PMID:24937763

  5. Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae

    SciTech Connect

    Ito, Keisuke; Sugawara, Taishi; Shiroishi, Mitsunori; Tokuda, Natsuko; Kurokawa, Azusa; Misaka, Takumi; Makyio, Hisayoshi; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Nomura, Norimichi; Murata, Takeshi; Abe, Keiko; Iwata, So

    2008-07-11

    Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

  6. Selective methyl labeling of eukaryotic membrane proteins using cell-free expression.

    PubMed

    Linser, Rasmus; Gelev, Vladimir; Hagn, Franz; Arthanari, Haribabu; Hyberts, Sven G; Wagner, Gerhard

    2014-08-13

    Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins. PMID:24937763

  7. Time-dependent changes in antioxidative enzyme expression and photosynthetic activity of Chlamydomonas reinhardtii cells under acute exposure to cadmium and anthracene.

    PubMed

    Aksmann, Anna; Pokora, Wojciech; Baścik-Remisiewicz, Agnieszka; Dettlaff-Pokora, Agnieszka; Wielgomas, Bartosz; Dziadziuszko, Małgorzata; Tukaj, Zbigniew

    2014-12-01

    Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are present in the freshwater environment at concentrations that can be hazardous to the biota. Among HMs and PAHs, cadmium (Cd) and anthracene (ANT) are the most prevalent and toxic ones. The response of Chlamydomonas cells to Cd and ANT at concentrations that markedly reduced the growth of algal population was investigated in this study. At such concentrations, both cadmium and anthracene were recognized as oxidative stress inducers, since high concentration of H2O2 in treated cultures was observed. Therefore, as a part of the "molecular phase" of the cell response to this stress, we examined the time-dependent expression of genes encoding the main antioxidative enzymes: superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as the activity of these enzymes in cells, with special attention paid to chloroplastic and mitochondrial isoforms of SOD. To characterize the cell response at the "physiological level", we examined the photosynthetic activity of stressed cells via analysis of chlorophyll a fluorescence in vivo. In contrast to standard ecotoxicity studies in which the growth end-points are usually determined, herein we present time-dependent changes in algal cell response to Cd- and ANT-induced stress. The most significant effect(s) of the toxicants on photosynthetic activity was observed in the 6th hour, when strong depression of PI parameter value, an over 50 percent reduction of the active reaction center fraction (RC0) and a 3-fold increase in non-photochemical energy dissipation (DI0/RC) were noted. At the same time, the increase (up to 2.5-fold) in mRNA transcript of SOD and CAT genes, followed by the enhancement in the enzyme activity was observed. The high expression of the Msd 3 gene in treated Chlamydomonas cells probably complements the partial loss of chloroplast Fe-SOD and APX activity, while catalase and Mn-SOD 5 seem to be the major enzymes responsible for

  8. Bemisia tabaci-infested tomato plants show a phase-specific pattern of photosynthetic gene expression indicative of disrupted electron flow leading to photo-oxidation and plant death

    PubMed Central

    Estrada-Hernández, María Gloria

    2009-01-01

    A suppression-subtractive-hybridization (SSH) strategy led to the identification of several genes whose expression was differentially modified in response to different larval phases present during the infestation process of tomato plants (Solanum lycopersicum) by virus-free whitefly Bemisia tabaci (Bt). The findings regarding photosynthetic gene expression were in accordance to previous studies reporting altered patterns of expression as a result of insect herbivory. However, the examination, in this study, of four stages of larval Bt development permitted the detection of phase-dependent changes in gene expression which appeared to target specific photosynthetic complexes. Thus, an upregulation of photosystem II genes in the latter two phases of Bt development contrasted with a general repression of genes belonging to the three other photosynthetic complexes, in addition to a number of genes coding for proteins associated with the oxygen evolving complex and the Calvin cycle. We propose that the contrasting pattern of expression led to an over-excitation of PSII and consequent oxidative damage, as suggested by the concomitant upregulation of oxidative stress genes, and could have contributed to the wide-spread necrosis observed in Bt-infested tomato plants at late stages of the plant-insect interaction. PMID:19826216

  9. Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

    NASA Astrophysics Data System (ADS)

    Miledi, Ricardo; Eusebi, Fabrizio; Martínez-Torres, Ataúlfo; Palma, Eleonora; Trettel, Flavia

    2002-10-01

    The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to -aminobutyric acid, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.

  10. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    PubMed Central

    Ooi, Amanda; Wong, Aloysius; Esau, Luke; Lemtiri-Chlieh, Fouad; Gehring, Chris

    2016-01-01

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins. PMID:27486406

  11. Membrane-targeted HrpNEa can modulate apple defense gene expression.

    PubMed

    Vergne, E; de Bernonville, T Dugé; Dupuis, F; Sourice, S; Cournol, R; Berthelot, P; Barny, M A; Brisset, M N; Chevreau, E

    2014-02-01

    Fire blight caused by Erwinia amylovora is the major bacterial disease of tribe Maleae, including apple. Among the proteins secreted by this bacterium, HrpNEa, also called harpin, is known to induce hypersensitive response in nonhost plants and to form amyloid oligomers leading to pore opening in the plasma membrane and alteration of membrane homeostasis. To better understand the physiological effects of HrpNEa in the host plant, we produced transgenic apple plants expressing HrpNEa with or without a secretion signal peptide (SP). HrpNEa expressed with a SP was found to be associated within the membrane fraction, in accordance with amyloidogenic properties and the presence of transmembrane domains revealed by in silico analysis. Expression analysis of 28 apple defense-related genes revealed gene modulations in the transgenic line expressing membrane-targeted HrpNEa. While apple transgenic trees displaying a high constitutive expression level of SP-HrpNEa showed a slight reduction of infection frequency after E. amylovora inoculation, there was no decrease in the disease severity. Thus HrpNEa seems to act as an elicitor of host defenses, when localized in the host membrane. PMID:24156770

  12. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  13. The Photosynthetic Apparatus and Its Regulation in the Aerobic Gammaproteobacterium Congregibacter litoralis gen. nov., sp. nov

    PubMed Central

    Spring, Stefan; Lünsdorf, Heinrich; Fuchs, Bernhard M.; Tindall, Brian J.

    2009-01-01

    Background There is accumulating evidence that in some marine environments aerobic bacteriochlorophyll a-producing bacteria represent a significant part of the microbial population. The interaction of photosynthesis and carbon metabolism in these interesting bacteria is still largely unknown and requires further investigation in order to estimate their contribution to the marine carbon cycle. Methodology/Principal Findings Here, we analyzed the structure, composition and regulation of the photosynthetic apparatus in the obligately aerobic marine gammaproteobacterium KT71T. Photoheterotrophically grown cells were characterized by a poorly developed lamellar intracytoplasmic membrane system, a type 1 light-harvesting antenna complex and a photosynthetic reaction center associated with a tetraheme cytochrome c. The only photosynthetic pigments produced were bacteriochlorophyll a and spirilloxanthin. Under semiaerobic conditions KT71T cells expressing a photosynthetic apparatus showed a light-dependent increase of growth yield in the range of 1.3–2.5 fold. The expression level of the photosynthetic apparatus depended largely on the utilized substrate, the intermediary carbon metabolism and oxygen tension. In addition, pigment synthesis was strongly influenced by light, with blue light exerting the most significant effect, implicating that proteins containing a BLUF domain may be involved in regulation of the photosynthetic apparatus. Several phenotypic traits in KT71T could be identified that correlated with the assumed redox state of growing cells and thus could be used to monitor the cellular redox state under various incubation conditions. Conclusions/Significance In a hypothetical model that explains the regulation of the photosynthetic apparatus in strain KT71T we propose that the expression of photosynthesis genes depends on the cellular redox state and is maximal under conditions that allow a balanced membrane redox state. So far, bacteria capable of an

  14. Expression and localization of predicted inclusion membrane proteins in Chlamydia trachomatis.

    PubMed

    Weber, Mary M; Bauler, Laura D; Lam, Jennifer; Hackstadt, Ted

    2015-12-01

    Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a membrane-bound vacuole termed the inclusion. Early in the infection cycle, the pathogen extensively modifies the inclusion membrane through incorporation of numerous type III secreted effector proteins, called inclusion membrane proteins (Incs). These proteins are characterized by a bilobed hydrophobic domain of 40 amino acids. The presence of this domain has been used to predict up to 59 putative Incs for C. trachomatis; however, localization to the inclusion membrane with specific antibodies has been demonstrated for only about half of them. Here, we employed recently developed genetic tools to verify the localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of epitope-tagged putative Incs identified 10 that were previously unverified as inclusion membrane localized and thus authentic Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by colocalization with phosphorylated Src (p-Src). Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. Using Yersinia as a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they may not be true Incs. Collectively, our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will advance our understanding of how the pathogen maintains its unique intracellular niche and mediates interactions with the host. PMID:26416906

  15. Tumor necrosis factor-alpha is expressed by glomerular visceral epithelial cells in human membranous nephropathy.

    PubMed Central

    Neale, T. J.; Rüger, B. M.; Macaulay, H.; Dunbar, P. R.; Hasan, Q.; Bourke, A.; Murray-McIntosh, R. P.; Kitching, A. R.

    1995-01-01

    The role of tumor necrosis factor alpha (TNF-alpha) was examined in biopsy-proven glomerulonephritis by immunohistochemistry, in situ hybridization, immunogold electron microscopy, immunoassay in serum and urine, and urinary immunoblot. Striking glomerular capillary wall and visceral glomerular epithelial cell TNF-alpha protein staining was observed in all cases of membranous nephropathy and membranous lupus nephropathy. Staining was less frequently observed in crescentic glomerulonephritis and in isolated cases of other histological subtypes of glomerulonephritis, usually in association with glomerular macrophages. By immunogold electron microscopy TNF-alpha was localized in membranous nephropathy within the visceral glomerular epithelial cells, and also in the glomerular basement membrane, especially in relation to immune deposits. In situ hybridization localized TNF-alpha mRNA exclusively to glomerular epithelial cells in all biopsies with membranous morphology but not in other histological subtypes. Concentrations of TNF-alpha were significantly increased compared with normal controls in the urine of patients with membranous nephropathy and with crescentic glomerulonephritis. The expression of TNF-alpha by glomerular epithelial cells exclusively and universally in biopsies showing a membranous morphology strongly suggests this cytokine has a role in the pathogenesis of membranous nephropathy. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:7778683

  16. Expression and purification of membrane protein diacylglycerol acyltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG. Over-expression of DGATs increases TAG in seeds and other tissues. DGAT knockout mice are resista...

  17. In-Situ Observation of Membrane Protein Folding during Cell-Free Expression

    PubMed Central

    Fitter, Jörg; Büldt, Georg; Heberle, Joachim; Schlesinger, Ramona; Ataka, Kenichi

    2016-01-01

    Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution. PMID:26978519

  18. Tissue specificity of a baculovirus expressed, basement membrane-degrading protease in larvae of Heliothis virescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ScathL is a cathepsin L-like cysteine protease from flesh fly Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm, Heliothis virescens, significantly faste...

  19. Photosynthetic reaction center complexes from heliobacteria

    NASA Technical Reports Server (NTRS)

    Trost, J. T.; Vermaas, W. F. J.; Blankenship, R. E.

    1991-01-01

    Photosynthetic reaction centers are pigment-protein complexes that are responsible for the transduction of light energy into chemical energy. Considerable evidence indicates that photosynthetic organisms were present very early in the evolution of life on Earth. The goal of this project is to understand the early evolutionary development of photosynthesis by examining the properties of reaction centers isolated from certain contemporary organisms that appear to contain the simplest photosynthetic reaction centers. The major focus is on the family of newly discovered strictly anaerobic photosynthetic organisms that are grouped with the gram-positive phylum of bacteria. The properties of these reactions centers suggest that they may be the descendants of an ancestor that also gave rise to Photosystem 1 found in oxygen-evolving photosynthetic organisms. Photoactive reaction center-core antenna complexes were isolated from the photosynthetic bacteria, Heliobacillus mobilis and Heliobacterium gestii, by extraction of membranes with Deriphat 160C followed by differential centrifugation and sucrose density gradient centrifugation. Other aspects of this investigation are briefly discussed.

  20. Feasibility of a photosynthetic artificial lung.

    PubMed

    Basu-Dutt, S; Fandino, M R; Salley, S O; Thompson, I M; Whittlesey, G C; Klein, M D

    1997-01-01

    The success of extracorporeal membrane oxygenation (ECMO) for the treatment of acute respiratory failure has led to consideration of the development of a more portable, and perhaps even implantable, artificial lung. The authors suggest a bioregenerative life support system that includes a photo-synthetic organism that can remove CO2 and produce O2 in the presence of an energy source. To build a model of such a photosynthetic artificial lung, the photosynthetic capability of a high temperature strain of the algae Chlorella pyrenoidosa was maximized at a cell density of 25 million cells/ml to serve as the O2 producer and CO2 remover. The "patient" in this model was comprised of 1 L of medium or 350 ml of blood, interfaced with the photosynthetic system across a gas transfer membrane. The experiments demonstrated the ability of the plant cells to supply O2 and remove CO2 from the "patient" with a maximum rate of 0.55 mmoles/L/hr under the most favorable measured operating conditions. The projected rate of 1.0 mmoles/L/hr required for physiologic applications is not totally ab absurd idea, with a slightly modified set-up. Modifications may be in the form of regulating the photosynthetic pathway or genetically engineering a hybrid strain with enhanced O2 producing and suppressed photoinhibition capacity. PMID:9242940

  1. 1-methylcyclopropene (1-MCP)-induced alteration in leaf photosynthetic rate, chlorophyll fluorescence, respiration and membrane damage in rice (Oryza sativa L.) under high night temperature

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High night temperature (HNT) can induce ethylene-triggered reactive oxygen species production, which can cause premature leaf senescence and membrane damage, thereby affecting production, consumption and transfer of photosynthates, and yield. The 1-methylcyclopropene (1-MCP) can competitively bind w...

  2. Small-Scale Screening to Large-Scale Over-Expression of Human Membrane Proteins for Structural Studies.

    PubMed

    Chaudhary, Sarika; Saha, Sukanya; Thamminana, Sobrahani; Stroud, Robert M

    2016-01-01

    Membrane protein structural studies are frequently hampered by poor expression. The low natural abundance of these proteins implies a need for utilizing different heterologous expression systems. E. coli and yeast are commonly used expression systems due to rapid cell growth at high cell density, economical production, and ease of manipulation. Here we report a simplified, systematically developed robust strategy from small-scale screening to large-scale over-expression of human integral membrane proteins in the mammalian expression system for structural studies. This methodology streamlines small-scale screening of several different constructs utilizing fluorescence size-exclusion chromatography (FSEC) towards optimization of buffer, additives, and detergents for achieving stability and homogeneity. This is followed by the generation of stable clonal cell lines expressing desired constructs, and lastly large-scale expression for crystallization. These techniques are designed to rapidly advance the structural studies of eukaryotic integral membrane proteins including that of human membrane proteins. PMID:27485338

  3. Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

    PubMed Central

    Iruela-Arispe, M L; Lane, T F; Redmond, D; Reilly, M; Bolender, R P; Kavanagh, T J; Sage, E H

    1995-01-01

    SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that

  4. Cloning of immunodominant membrane protein genes of phytoplasmas and their in planta expression.

    PubMed

    Kakizawa, Shigeyuki; Oshima, Kenro; Ishii, Yoshiko; Hoshi, Ayaka; Maejima, Kensaku; Jung, Hee-Young; Yamaji, Yasuyuki; Namba, Shigetou

    2009-04-01

    Phytoplasmas are plant pathogenic bacteria that cause devastating yield losses in diverse crops worldwide. Although the understanding of the pathogen biology is important in agriculture, the inability to culture phytoplasmas has hindered their full characterization. Previous studies demonstrated that immunodominant membrane proteins could be classified into three types, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp), and they are nonhomologous to each other. Here, cloning and sequencing of imp-containing genomic fragments were performed for several groups of phytoplasma including the aster yellows and rice yellow dwarf groups, for which an imp sequence has not previously been reported. Sequence comparison analysis revealed that Imps are highly variable among phytoplasmas, and clear positive selection was observed in several Imps, suggesting that Imp has important roles in host-phytoplasma interactions. As onion yellows (OY) phytoplasma was known to have Amp as the immunodominant membrane protein, the protein accumulation level of Imp in planta was measured compared with that of Amp. The resulting accumulation of Imp was calculated as approximately one-tenth that of Amp, being consistent with the immunodominant property of Amp in OY. It is suggested that an ancestral type of immunodominant membrane protein could be Imp, and subsequently the expression level of Amp or IdpA is increased in several phytoplasma groups. PMID:19222574

  5. Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

    PubMed Central

    Mourot, Brigitte; Nguyen, Thaovi; Fostier, Alexis; Bobe, Julien

    2006-01-01

    Background In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. Methods In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. Results Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. Conclusion Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels. PMID:16457725

  6. Photosynthetic water splitting

    SciTech Connect

    Greenbaum, E.

    1981-01-01

    The photosynthetic unit of hydrogen evolution, the turnover time of photosynthetic hydrogen production, and hydrogenic photosynthesis are discussed in the section on previous work. Recent results are given on simultaneous photoproduction of hydrogen and oxygen, kinetic studies, microscopic marine algae-seaweeds, and oxygen profiles.

  7. Role of membrane depolarization and extracellular calcium in increased complement receptor expression during neutrophil (PMN) activation

    SciTech Connect

    Berger, M.; Wetzler, E.; Birx, D.L.

    1986-03-05

    During PMN activation the surface expression of receptors (R) for C3b and C3bi increases rapidly. This is necessary for optimal cell adhesion, migration, and phagocytosis. Following stimulation with fMLP or LTB-4, the increased expression of C3bR depends only on the Ca/sup + +/ released from intracellular stores and is not inhibited by 5mM EDTA, while the increase in C3biR also requires extracellular Ca/sup + +/. CR expression also increases when the PMN are depolarized with 140 mM K/sup +/, but with this stimulus, EDTA inhibits C3bR by 67% and C3biR 100%, suggesting that intracellular Ca/sup + +/ stores may not be released. Pertussis toxin caused dose-dependent inhibition of both CR responses to fMLP and also inhibited the increases in both CR induced by K/sup +/. Membrane depolarization (monitored by di-O-C5 fluorescence) due to fMLP was similarly inhibited by toxin but the depolarization due to K/sup +/ was not. The dose of phorbol myristate acetate that maximally increased CR expression, 0.1 ng/ml, did not depolarize the membrane. These results suggest that membrane depolarization is neither necessary nor sufficient for increased CR expression. A Ca/sup + +/ and GTP binding protein-dependent enzyme such as phospholipase C is necessary to the amplify initial signals generated either by release of Ca/sup + +/ stores or by opening voltage dependent Ca/sup + +/ channels following membrane depolarization.

  8. Tat Peptide-Mediated Soluble Expression of the Membrane Protein LSECtin-CRD in Escherichia coli

    PubMed Central

    Dong, Guofu; Wang, Changzhen; Wu, Yonghong; Cong, Jianbo; Cheng, Li; Wang, Mingqun; Zhao, Pengkai; Tang, Li; Zhang, Chenggang; Wu, Ke

    2013-01-01

    The human liver and lymph node sinusoidal endothelial cell C-type lectin (hLSECtin), a type II integral membrane protein, containing a Ca2+-dependent carbohydrate recognition domain (CRD), has a well-established biological activity, yet its three-dimensional structure is unknown due to low expression yields and aggregation into inclusion bodies. Previous study has demonstrated that the HIV-1 virus-encoded Tat peptide (‘YGRKKRRQRRR’) can increase the yields and the solubility of heterologous proteins. However, whether the Tat peptide could promote the high-yield and soluble expression of membrane proteins in Escherichia coli is not known. Therefore, the prokaryotic expression vector pET28b-Tat-hLSECtin-CRD (using pET28b and pET28b-hLSECtin-CRD as controls) was constructed, and transformed into E. coli BL21 (DE3) cells and induced with isopropyl-β-d-thiogalactoside (IPTG) followed with identifying by SDS-PAGE and Western blot. Subsequently, the bacterial subcellular structure, in which overexpressed the heterologous proteins Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD, was analyzed by transmission electron microscope (TEM) respectively, and the mannose-binding activity of Tat-hLSECtin-CRD was also determined. Expectedly, the solubility of Tat-LSECtin-CRD significantly increased compared to Tat-free LSECtin-CRD (**p < 0.01) with prolonged time, and the Tat-LSECtin-CRD had a significant mannose-binding activity. The subcellular structure analysis indicated that the bacterial cells overexpressed Tat-hLSECtin-CRD exhibited denser region compared with controls, while dot denser region aggregated in the two ends of bacterial cells overexpressed Tat-free hLSECtin-CRD. This study provided a novel method for improving the soluble expression of membrane proteins in prokaryotic systems by fusion with the Tat peptide, which may be potentially expanded to the expression of other membrane proteins. PMID:24358298

  9. Process for photosynthetically splitting water

    SciTech Connect

    Greenbaum, E.

    1982-01-28

    In one form of the invention, hydrogen is produced by providing a reactor containing a body of water. The water contains photolytic material, i.e., photoactive material containing a hydrogen-catalyst. The interior of the reactor is isolated from atmosphere and includes a volume for receiving gases evolved from the body of water. The photolytic material is exposed to light to effect photosynthetic splitting of the water into gaseous hydrogen and oxygen. The gas-receiving volume is continuously evacuated by pumping to promote evolution of gaseous hydrogen and oxygen into that volume and to withdraw them therefrom. In another form of the invention, separation of the hydrogen and oxygen is effected by selectively diffusing the hydrogen through a heated semipermeable membrane in a separation zone while maintaining across the zone a magnetic field gradient biasing the oxygen away from the membrane. In a third form of the invention, the withdrawn gas is contacted with a membrane blocking flow of water vapor to the region for effecting recovery of the hydrogen. In a fourth embodiment, the invention comprises a process for selectively recovering hydrogen from a gas mixture comprising hydrogen and oxygen. The process is conducted in a separation zone and comprises contacting the mixture with a semipermeable membrane effecting selective diffusion of hydrogen while maintaining across the zone a magnetic field gradient effecting movement of oxygen in a direction away from the membrane.

  10. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    NASA Astrophysics Data System (ADS)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

    2011-06-01

    Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

  11. Tangled evolutionary processes with commonality and diversity in plastidial glycolipid synthesis in photosynthetic organisms.

    PubMed

    Hori, Koichi; Nobusawa, Takashi; Watanabe, Tei; Madoka, Yuka; Suzuki, Hideyuki; Shibata, Daisuke; Shimojima, Mie; Ohta, Hiroyuki

    2016-09-01

    In photosynthetic organisms, the photosynthetic membrane constitutes a scaffold for light-harvesting complexes and photosynthetic reaction centers. Three kinds of glycolipids, namely monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol, constitute approximately 80-90% of photosynthetic membrane lipids and are well conserved from tiny cyanobacteria to the leaves of huge trees. These glycolipids perform a wide variety of functions beyond biological membrane formation. In particular, the capability of adaptation to harsh environments through regulation of membrane glycolipid composition is essential for healthy growth and development of photosynthetic organisms. The genome analysis and functional genetics of the model seed plant Arabidopsis thaliana have yielded many new findings concerning the biosynthesis, regulation, and functions of glycolipids. Nevertheless, it remains to be clarified how the complex biosynthetic pathways and well-organized functions of glycolipids evolved in early and primitive photosynthetic organisms, such as cyanobacteria, to yield modern photosynthetic organisms like land plants. Recently, genome data for many photosynthetic organisms have been made available as the fruit of the rapid development of sequencing technology. We also have reported the draft genome sequence of the charophyte alga Klebsormidium flaccidum, which is an intermediate organism between green algae and land plants. Here, we performed a comprehensive phylogenic analysis of glycolipid biosynthesis genes in oxygenic photosynthetic organisms including K. flaccidum. Based on the results together with membrane lipid analysis of this alga, we discuss the evolution of glycolipid synthesis in photosynthetic organisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:27108062

  12. Use of a yeast-based membrane protein expression technology to overexpress drug resistance efflux pumps.

    PubMed

    Lamping, Erwin; Cannon, Richard D

    2010-01-01

    Azole antifungal drugs are used widely to treat people with oral fungal infections. Unfortunately, fungi can develop resistance to these drugs. This resistance can be due to the overexpression or mutation of cytochrome P450 14alpha-lanosterol demethylase, also known as ERG11 or CYP51, and/or the overexpression of membrane-located multidrug efflux pumps. We have developed a heterologous membrane protein expression system that can be used to study the structure and function of these proteins in the non-pathogenic, genetically stable, and versatile eukaryotic model organism, Saccharomyces cerevisiae. In this chapter we describe the techniques used to express the Candida albicans efflux pump Cdr1p in S. cerevisiae. PMID:20717788

  13. Overexpression of the Maize psbA Gene Enhances Drought Tolerance Through Regulating Antioxidant System, Photosynthetic Capability, and Stress Defense Gene Expression in Tobacco.

    PubMed

    Huo, Yongjin; Wang, Meiping; Wei, Yangyang; Xia, Zongliang

    2015-01-01

    The psbA (encoding D1 protein) plays an important role in protecting photosystem II (PSII) from oxidative damage in higher plants. In our previous study, the role of the psbA from maize (Zea mays. L) in response to SO2 stress was characterized. To date, information about the involvement of the psbA gene in drought response is scarce. Here we found that overexpression (OE) of ZmpsbA showed increased D1 protein abundance and enhanced drought stress tolerance in tobacco. The drought-tolerant phenotypes of the OE lines were accompanied by increases of key antioxidant enzymes SOD, CAT, and POD activities, but decreases of hydrogen peroxide, malondialdehyde, and ion leakage. Further investigation showed that the OE plants had much less reductions than the wild-type in the net photosynthesis rate (Pn), stomatal conductance (Gs), and the maximal photochemical efficiency of PSII (Fv/Fm) during drought stress; indicating that OE of ZmpsbA may alleviate photosynthesis inhibition during drought. qRT-PCR analysis revealed that there was significantly increased expression of NtLEA5, NtERD10C, NtAREB, and NtCDPK2 in ZmpsbA-OE lines. Together, our results indicate that ZmpsbA improves drought tolerance in tobacco possibly by alleviating photosynthesis reduction, reducing reactive oxygen species accumulation and membrane damage, and modulating stress defense gene expression. ZmpsbA could be exploited for engineering drought-tolerant plants in molecular breeding of crops. PMID:26793207

  14. Expression of two membrane fusion proteins, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, in choroid plexus epithelium.

    PubMed

    Chung, I; Burkart, A; Szmydynger-Chodobska, J; Dodd, K A; Trimble, W S; Miller, K V; Shim, M; Chodobski, A

    2003-01-01

    In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of

  15. Gene expression analysis of Drosophilaa Manf mutants reveals perturbations in membrane traffic and major metabolic changes

    PubMed Central

    2012-01-01

    Background MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf. Results The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the endoplasmic reticulum (ER) stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well. Conclusions We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome the UPR especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons. PMID:22494833

  16. Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions.

    PubMed

    Narayanan, Anoop; Ridilla, Marc; Yernool, Dinesh A

    2011-01-01

    A major rate-limiting step in determining structures of membrane proteins is heterologous protein production. Toxicity often associated with rapid overexpression results in reduced biomass along with low yields of target protein. Mitigation of toxic effects was achieved using a method we call "restrained expression," a controlled reduction in the frequency of transcription initiation by exploiting the infrequent transitions of Lac repressor to a free state from its complex with the lac-operator site within a T7lac promoter that occur in the absence of the inducer isopropyl β-D-1-thiogalactopyranoside. In addition, production of the T7 RNA polymerase that drives transcription of the target is limited using the tightly regulated arabinose promoter in Escherichia coli strain BL21-AI. Using this approach, we can achieve a 200-fold range of green fluorescent protein expression levels. Application to members of a family of ion pumps results in 5- to 25-fold increases in expression over the benchmark BL21(DE3) host strain. A viral ion channel highly toxic to E. coli can also be overexpressed. In comparative analyses, restrained expression outperforms commonly used E. coli expression strategies. The mechanism underlying improved target protein yield arises from minimization of protein aggregation and proteolysis that reduce membrane integrity and cell viability. This study establishes a method to overexpress toxic proteins. PMID:21031485

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

    PubMed

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

    2016-04-01

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

  18. Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

    SciTech Connect

    Rayter, Z. ); McIlhinney, R. ); Gusterson, B. )

    1989-08-01

    Con A acceptor glycoproteins were analyzed by 2D-PAGE and {sup 125}I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21, was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.

  19. Oxygen concentration inside a functioning photosynthetic cell.

    PubMed

    Kihara, Shigeharu; Hartzler, Daniel A; Savikhin, Sergei

    2014-05-01

    The excess oxygen concentration in the photosynthetic membranes of functioning oxygenic photosynthetic cells was estimated using classical diffusion theory combined with experimental data on oxygen production rates of cyanobacterial cells. The excess oxygen concentration within the plesiomorphic cyanobacterium Gloeobactor violaceus is only 0.025 μM, or four orders of magnitude lower than the oxygen concentration in air-saturated water. Such a low concentration suggests that the first oxygenic photosynthetic bacteria in solitary form could have evolved ∼2.8 billion years ago without special mechanisms to protect them against reactive oxygen species. These mechanisms instead could have been developed during the following ∼500 million years while the oxygen level in the Earth's atmosphere was slowly rising. Excess oxygen concentrations within individual cells of the apomorphic cyanobacteria Synechocystis and Synechococcus are 0.064 and 0.25 μM, respectively. These numbers suggest that intramembrane and intracellular proteins in isolated oxygenic photosynthetic cells are not subjected to excessively high oxygen levels. The situation is different for closely packed colonies of photosynthetic cells. Calculations show that the excess concentration within colonies that are ∼40 μm or larger in diameter can be comparable to the oxygen concentration in air-saturated water, suggesting that species forming colonies require protection against reactive oxygen species even in the absence of oxygen in the surrounding atmosphere. PMID:24806920

  20. Rab4GTPase modulates CFTR function by impairing channel expression at plasma membrane

    SciTech Connect

    Saxena, Sunil K. . E-mail: ssaxena@stevens.edu; Kaur, Simarna; George, Constantine

    2006-03-03

    Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein-protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein-protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function.

  1. Gene Expression Pattern of Cells From Inflamed and Normal Areas of Osteoarthritis Synovial Membrane

    PubMed Central

    Lambert, Cécile; Dubuc, Jean-Emile; Montell, Eulàlia; Vergés, Josep; Munaut, Carine; Noël, Agnès; Henrotin, Yves

    2014-01-01

    Objective To compare the gene expression patterns of synovial cells from inflamed or normal/reactive areas of synovial membrane obtained from the same patient with osteoarthritis (OA). Methods At the time of total knee replacement, synovial tissues were obtained from 12 patients with knee OA. The inflammation status of the synovial membrane was characterized according to macroscopic criteria and classified as normal/reactive or inflamed. Biopsy samples were cultured separately for 7 days. Microarray gene expression profiling was performed on normal/reactive and inflamed areas. Western blot and immunohistochemistry were used to confirm the identified genes that were differentially expressed. Results We identified 896 genes that were differentially expressed between normal/reactive and inflamed areas. The key pathways were related to inflammation, cartilage metabolism, Wnt signaling, and angiogenesis. In the inflammation network, the genes TREM1 and S100A9 were strongly up-regulated. The genes MMP3, MMP9, CTSH (cathepsin H), and CTSS (cathepsin S) were significantly up-regulated in the cartilage catabolism pathway, while the most up-regulated anabolism enzyme gene was HAS1. In the Wnt signaling pathway, the genes for Wnt-5a and low-density lipoprotein receptor–related protein 5 were up-regulated, while the gene FZD2 and the gene for Dkk-3 were down-regulated. Finally, STC1, which codes for a protein involved in angiogenesis, was identified as the most up-regulated gene in inflamed compared with normal/reactive areas. Conclusion This study is the first to identify different expression patterns between 2 areas of the synovial membrane from the same patient. These differences concern several key pathways involved in OA pathogenesis. This analysis also provides information regarding new genes and proteins as potential targets of treatment. PMID:24757147

  2. Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles*S⃞

    PubMed Central

    Cappuccio, Jenny A.; Blanchette, Craig D.; Sulchek, Todd A.; Arroyo, Erin S.; Kralj, Joel M.; Hinz, Angela K.; Kuhn, Edward A.; Chromy, Brett A.; Segelke, Brent W.; Rothschild, Kenneth J.; Fletcher, Julia E.; Katzen, Federico; Peterson, Todd C.; Kudlicki, Wieslaw A.; Bench, Graham; Hoeprich, Paul D.; Coleman, Matthew A.

    2008-01-01

    Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. PMID:18603642

  3. Expression of calcium transport proteins in the extraembryonic membranes of a viviparous snake, Virginia striatula.

    PubMed

    Fregoso, Santiago P; Stewart, James R; Ecay, Tom W

    2012-06-01

    Yolk is the primary source of calcium for embryonic growth and development for most squamates, irrespective of mode of parity. The calcified eggshell is a secondary source for embryonic calcium in all oviparous eggs, but this structure is lost in viviparous lineages. Virginia striatula is a viviparous snake in which embryos obtain calcium from both yolk and placental transport of uterine calcium secretions. The developmental pattern of embryonic calcium acquisition in V. striatula is similar to that for oviparous snakes. Calbindin-D(28K) is a marker for epithelial calcium transport activity and plasma membrane Ca(2+)-ATPase (PMCA) provides the energy to catalyze the final step in calcium transport. Expression of calbindin-D(28K) and PMCA was measured by immunoblotting in yolk sac splanchnopleure and chorioallantois of a developmental series of V. striatula to test the hypothesis that these proteins mediate calcium transport to embryos. In addition, we compared the expression of calbindin-D(28K) in extraembryonic membranes of V. striatula throughout development to a previously published expression pattern in an oviparous snake to test the hypothesis that the ontogeny of calcium transport function is independent of reproductive mode. Expression of calbindin-D(28K) increased in yolk sac splanchnopleure and chorioallantois coincident with calcium mobilization from yolk and uterine sources and with embryonic growth. The amount of PMCA in the chorioallantois did not change through development suggesting its expression is not rate limiting for calcium transport. The pattern of expression of calbindin-D(28K) and PMCA confirms our initial hypothesis that these proteins mediate embryonic calcium uptake. In addition, the developmental pattern of calbindin-D(28K) expression in V. striatula is similar to that of an oviparous snake, which suggests that calcium transport mechanisms and their regulation are independent of reproductive mode. PMID:22821861

  4. Candidate Gene and MicroRNA Expression in Fetal Membranes and Preterm Delivery Risk.

    PubMed

    Enquobahrie, Daniel A; Hensley, Mark; Qiu, Chunfang; Abetew, Dejene F; Hevner, Karin; Tadesse, Mahlet G; Williams, Michelle A

    2016-06-01

    We investigated candidate gene and microRNA (miRNA) expression in amnion and chorion in relation to risk of preterm delivery (PTD). Amnion and chorion were separated from placenta and collected at delivery from participants who delivered at term (N = 10) and from participants who delivered preterm following spontaneous labor (sPTL-PTD; N = 10), premature rupture of membranes (PPROM-PTD; N = 10), and preeclampsia (PE-PTD; N = 10). Expression of genes (metalloproteinase [MMP] 2, MMP-9, and tissue inhibitors of MMP-1) and miRNAs (miR-199a*, -202*, -210, -214, -223, and -338) was profiled using quantitative real-time polymerase chain reaction approaches. Adjusted multinomial logistic regression models were used to calculate relative risk ratios (RRR), 95% confidence intervals, and P values. Among controls, the expression of miR-199a*, -202*, and -214 was lower in the amnion compared with their expression in the chorion, whereas the expression of miR-210 was higher in the amnion compared with its expression in the chorion (all P values < .05). In the amnion, MMP-9 expression was associated with PTD risk (overall P value = .0092), and MMP-9 expression was positively associated with the risk of PPROM-PTD (RRR: 31.10) and inversely associated with the risk of PE-PTD (RRR:6.55e-6), although individual associations were not statistically significant. In addition, in the amnion, the expression of miR-210 (RRR: 0.45; overall P value = .0039) was inversely associated with the risk of PE-PTD, and miR-223 was inversely associated with all subtypes of PTD (overall P value = .0400). The amnion and chorion differ in their miRNA expression. The expression of MMP-9, miR-210, and -223 in the amnion is associated with PTD risk. PMID:26507872

  5. LAPTM5: A novel lysosomal-associated multispanning membrane protein preferentially expressed in hematopoietic cells

    SciTech Connect

    Adra, C.N.; Zhu, Shaochun; Ko, Jone-Long

    1996-07-15

    While a large body of knowledge about cell membrane proteins exists, much less is known about the repertoire and function of integral membrane proteins of intracellular organelles. In looking for novel classes of genes that are functionally important to hematopoietic cells, we have cloned the cDNA for a gene preferentially expressed in adult hematopoietic tissues. During embryonic development the gene is expressed in both hematopoietic and nonhematopoietic tissues. In cell lines the gene is expressed specifically in hematopoietic lineages, whereas in normal adult tissues the mRNA is preferentially detected at high levels in lymphoid and myeloid tissues. The predicted protein is a pentaspanner with no homology to known genes and conserved across evolution. Immunocytological and cell fractionation studies with a specific antibody revealed a protein localizing in lysosomes. The gene, provisionally named LAPTM5, maps to chromosome 1p34. The expression pattern of the gene together with preliminary evidence that the protein interacts with ubiquitin indicates that the protein may have a special functional role during embryogenesis and in adult hematopoietic cells. 53 refs., 9 figs.

  6. Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

    PubMed

    Boggavarapu, Rajendra; Hirschi, Stephan; Harder, Daniel; Meury, Marcel; Ucurum, Zöhre; Bergeron, Marc J; Fotiadis, Dimitrios

    2016-01-01

    This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail. PMID:27485339

  7. Expression of recombinant dystrophin and its localization to the cell membrane.

    PubMed

    Lee, C C; Pearlman, J A; Chamberlain, J S; Caskey, C T

    1991-01-24

    Duchenne's muscular dystrophy (DMD) is an X-linked progressive myopathy caused by a defect in the DMD gene locus. The gene corresponding to the DMD locus produces a 14-kilobase (kb) messenger RNA that codes for a large cytoskeletal membrane protein, dystrophin. DMD and Becker's muscular dystrophy are the consequences of dystrophin mutations. The exact biological function of dystrophin remains unknown but it has been demonstrated that it is localized to the cytoplasmic face of the cell membrane and has direct interaction with several other membrane proteins. We report here the synthesis of a 14-kb full-length complementary DNA for the mouse muscle dystrophin mRNA and the expression of this cDNA in COS cells. The recombinant dystrophin is indistinguishable from mouse muscle dystrophin by western blot analysis with anti-dystrophin antibodies and was shown by an immunofluorescent technique to be localized in the cell membrane. Our successful construction of a functional full-length cDNA opens opportunities for the study of structure and function of dystrophin and provides an opportunity to initiate gene therapy studies. PMID:1824797

  8. Expression of the axonal membrane glycoprotein M6a is regulated by chronic stress.

    PubMed

    Cooper, Ben; Fuchs, Eberhard; Flügge, Gabriele

    2009-01-01

    It has been repeatedly shown that chronic stress changes dendrites, spines and modulates expression of synaptic molecules. These effects all may impair information transfer between neurons. The present study shows that chronic stress also regulates expression of M6a, a glycoprotein which is localised in axonal membranes. We have previously demonstrated that M6a is a component of glutamatergic axons. The present data reveal that it is the splice variant M6a-Ib, not M6a-Ia, which is strongly expressed in the brain. Chronic stress in male rats (3 weeks daily restraint) has regional effects: quantitative in situ hybridization demonstrated that M6a-Ib mRNA in dentate gyrus granule neurons and in CA3 pyramidal neurons is downregulated, whereas M6a-Ib mRNA in the medial prefrontal cortex is upregulated by chronic stress. This is the first study showing that expression of an axonal membrane molecule is differentially affected by stress in a region-dependent manner. Therefore, one may speculate that diminished expression of the glycoprotein in the hippocampus leads to altered output in the corresponding cortical projection areas. Enhanced M6a-Ib expression in the medial prefrontal cortex (in areas prelimbic and infralimbic cortex) might be interpreted as a compensatory mechanism in response to changes in axonal projections from the hippocampus. Our findings provide evidence that in addition to alterations in dendrites and spines chronic stress also changes the integrity of axons and may thus impair information transfer even between distant brain regions. PMID:19180239

  9. The γ3 Chain of Laminin is Widely But Differentially Expressed in Murine Basement Membranes: Expression and Functional Studies

    PubMed Central

    Li, Yong N.; Radner, Stephanie; French, Margaret M.; Pinzón-Duarte, Germán; Daly, Gerard H.; Burgeson, Robert E.; Koch, Manuel; Brunken, William J.

    2012-01-01

    Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an α, a β and a γ chain. Previous studies have shown that the γ3 chain is partnered with either the β1 chain (in placenta) or β2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the γ3 chain is expressed in both apical and basal compartments (Gersdorff et al., 2005; Koch et al., 1999; Yan and Cheng, 2006). This study investigates the expression pattern of the γ3 chain in mouse. We developed three new γ3-reactive antibodies, and we show that the γ3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the γ1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where γ3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the γ3 chain in mice; unlike other laminin null mice (α5, β2, γ1 nulls) (Miner et al., 1998; Noakes et al., 1995; Smyth et al., 1999), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the γ3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system. PMID:22222602

  10. Progesterone, Inflammatory Cytokine (TNF-α), and Oxidative Stress (H2O2) Regulate Progesterone Receptor Membrane Component 1 Expression in Fetal Membrane Cells.

    PubMed

    Meng, Yan; Murtha, Amy P; Feng, Liping

    2016-09-01

    Progesterone receptor membrane component 1 (PGRMC1) is an important novel mediator of progesterone (P4) function in fetal membrane cells. We demonstrated previously that PGRMC1 is differentially expressed in fetal membranes among pregnancy subjects and diminished in preterm premature rupture of membrane subjects. In the current study, we aim to elucidate whether PGRMC1 expression is regulated by P4, tumor necrosis factor α (TNF-α), and H2O2 in fetal membrane cells. Primary cultured membrane cells were serum starved for 24 hours followed by treatments of P4, 17 hydroxyprogesterone caproate, and medroxyprogesterone 17 acetate (MPA) at 10(-7) mol/L with ethanol as vehicle control; TNF-α at 10, 20, and 50 ng/mL with phosphate-buffered saline (PBS) as control; and H2O2 at 10 and 100 μmol/L with culture media as control for 24, 48, and 72 hours. The messenger RNA (mRNA) and protein expression of PGRMC1 was quantified using polymerase chain reaction and Western blotting, respectively. We found that PGRMC1 protein expression was regulated by MPA, TNF-α, and H2O2 in a dose-dependent manner. This regulation is also specific to the type of cell (amnion, chorion, or decidua). The upregulation of PGRMC1 by MPA might be mediated through glucocorticoid receptor (GR) demonstrated using amnion and chorion cells model with GR knockdown by specific small interfering RNA transfection. The mRNA expression of PGRMC1 was decreased by H2O2 (100 μmol/L) treatment in amnion cells, which might ultimately result in downregulation of PGRMC1 protein as our data demonstrated. None of other treatments changed PGRMC1 mRNA level in these cells. We conclude that these stimuli act as regulatory factors of PGRMC1 in a cell-specific manner. PMID:26919974

  11. Differential Regulation of Membrane CD14 Expression and Endotoxin-Tolerance in Alveolar Macrophages

    PubMed Central

    Lin, Shu-Min; Frevert, Charles W.; Kajikawa, Osamu; Wurfel, Mark M.; Ballman, Kimberly; Mongovin, Stephen; Wong, Venus A.; Selk, Amy; Martin, Thomas R.

    2014-01-01

    SUMMARY CD14 is important in the clearance of bacterial pathogens from lungs. However, the mechanisms that regulate the expression of membrane CD14 (mCD14) on alveolar macrophages (AM) have not been studied in detail. This study examines the regulation of mCD14 on AM exposed to Escherichia coli in vivo and in vitro and explores the consequences of changes in mCD14 expression. The expression of mCD14 was decreased on AM exposed to E. coli in vivo and AM incubated with lipopolysaccharide (LPS) or E. coli in vitro. Polymyxin B abolished LPS effects but only partially blocked the effects of E. coli. Blockade of extracellular signal-regulated kinase pathways attenuated LPS and E. coli-induced decrease in mCD14 expression. Inhibition of proteases abrogated the LPS-induced decrease in mCD14 expression on AM and the release of sCD14 into the supernatants, but did not affect the response to E. coli. The production of TNF-α in response to a second challenge with Staphylococcus aureus or zymosan was decreased in AM following incubation with E. coli but not LPS. These studies show that distinct mechanisms regulate the expression of mCD14 and the induction of endotoxin-tolerance in AM and suggest that AM function is impaired at sites of bacterial infection. PMID:15059784

  12. Erythroid 5-aminolevulinate synthase mediates the upregulation of membrane band 3 protein expression by iron.

    PubMed

    Huang, Qianchuan; Li, Jinying; Feng, Weihua; Xu, Yanqun; Huang, Zhenxia; Lv, Shuqing; Zhou, Hong; Gao, Lei

    2010-03-01

    Iron deficiency leads to abnormal expression and function of band 3 protein in erythrocytes, but the underlying mechanisms remain elusive. The mRNA of erythroid-specific 5-aminolevulinate synthase (eALAS) contains an iron response element and the eALAS protein is an important mediator of iron utilization by erythrocytes. In this study, we investigated the effect of short hairpin RNA (shRNA) mediated silencing of eALAS on the expression of band 3 protein induced by iron. By real-time RT-PCR and Western blot we showed that at mRNA and protein level iron-induced expression of band 3 protein was lower in eALAS-shRNA transfected K562 cells than in control cells. Of note, the lowest expression was detected in K562 cells cultured in iron deficiency condition (p < 0.01). Thus either iron deficiency or depletion of eALAS could suppress the expression of erythroid band 3 protein. These results demonstrated for the first time that iron and the iron-regulatory system regulate the expression of the erythrocyte membrane proteins. PMID:20087844

  13. Susceptibility of Pseudomonas aeruginosa isolates to ceftazidime is unrelated to the expression of the outer membrane protein OprC.

    PubMed

    Pérez, F J; Navarro, D; Gimeno, C; García-de-Lomas, J

    1997-01-01

    Previously, it has been postulated that the porin OprC facilitates the diffusion of ceftazidime through the outer membrane of Pseudomonas aeruginosa. To further investigate this claim, the outer membrane protein (OMP) profiles of 22 ceftazidime-susceptible clinical isolates were analyzed. No correlation was found between MIC values and the level of expression of OprC. Further, OprC was either undetectable or expressed in reduced amounts in 12 isolates. In contrast, OprF and OprE were present in all isolates studied. This study suggests that OprC is dispensable for the permeation of ceftazidime through the outer membrane of P. aeruginosa. PMID:8996738

  14. Proteomic analysis of Lawsonia intracellularis reveals expression of outer membrane proteins during infection.

    PubMed

    Watson, Eleanor; Alberdi, M Pilar; Inglis, Neil F; Lainson, Alex; Porter, Megan E; Manson, Erin; Imrie, Lisa; Mclean, Kevin; Smith, David G E

    2014-12-01

    Lawsonia intracellularis is the aetiological agent of the commercially significant porcine disease, proliferative enteropathy. Current understanding of host-pathogen interaction is limited due to the fastidious microaerophilic obligate intracellular nature of the bacterium. In the present study, expression of bacterial proteins during infection was investigated using a mass spectrometry approach. LC-ESI-MS/MS analysis of two isolates of L. intracellularis from heavily-infected epithelial cell cultures and database mining using fully annotated L. intracellularis genome sequences identified 19 proteins. According to the Clusters of Orthologous Groups (COG) functional classification, proteins were identified with roles in cell metabolism, protein synthesis and oxidative stress protection; seven proteins with putative or unknown function were also identified. Detailed bioinformatic analyses of five uncharacterised proteins, which were expressed by both isolates, identified domains and motifs common to other outer membrane-associated proteins with important roles in pathogenesis including adherence and invasion. Analysis of recombinant proteins on Western blots using immune sera from L. intracellularis-infected pigs identified two proteins, LI0841 and LI0902 as antigenic. The detection of five outer membrane proteins expressed during infection, including two antigenic proteins, demonstrates the potential of this approach to interrogate L. intracellularis host-pathogen interactions and identify novel targets which may be exploited in disease control. PMID:25457368

  15. Expression, localisation and phylogeny of a novel family of plant-specific membrane proteins.

    PubMed

    Kasaras, A; Kunze, R

    2010-09-01

    In a screen for senescence-associated genes in Arabidopsis thaliana, a novel, highly up-regulated membrane protein was identified. It is a member of an uncharacterised, strictly plant-specific gene family and was named AtDMP1 (Arabidopsis thaliana DUF679 domain membrane protein 1). The AtDMP proteins are predicted to have four transmembrane spans, with cytosolic amino- and carboxy-termini. In this study, we investigated the phylogenetic distribution of DMP proteins, their tissue-specific expression and subcellular localisation in A. thaliana. The Chlamydomonas reinhardtii and Physcomitrella patens genomes in dicots contain only a single DMP gene copy, whereas there are five to 13 DMP genes and 11-16 in monocots, many of which supposedly result from recent gene duplications. The ubiquitous occurrence of DMP proteins in green plants and their absence from other kingdoms suggest a role in plant-specific processes. In A. thaliana, expression of nine out of ten DMP genes was detected. The expression patterns were found to be markedly tissue- and development-specific; thus, functional redundancy of most proteins is unlikely. The occurrence of several AtDMPs in tissues undergoing senescence (AtDMP1, -3, -4), dehiscence (AtDMP7) or abscission (AtDMP2, -4, -7) suggests involvement of DMPs in different types of programmed cell death. AtDMP-eGFP fusion proteins were found to localise either to the endoplasmic reticulum, the tonoplast or, under certain conditions, to both membrane systems. Further investigations are in progress to elucidate functions of the AtDMP proteins. PMID:20712629

  16. A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1 Acetylcholine Receptors Affects Plasma Membrane Expression

    PubMed Central

    Ehlert, Frederick J.; Shults, Crystal A.

    2010-01-01

    We investigated the functional role of a conserved motif, F(x)6LL, in the membrane proximal C-tail of the human muscarinic M1 (hM1) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence 423FRDTFRLLL431. Wild-type and mutant hM1 receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [3H]N-methylscopolamine binding assays. The plasma membrane expression of hM1 receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM1 receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L430 and L431 play a role in folding hM1 receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V46 and L47, that, when mutated, reduce the plasma membrane expression of hM1 receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM1 receptors to achieve a transport-competent state. PMID:19841475

  17. Prostate-specific membrane antigen protein expression in tumor tissue and risk of lethal prostate cancer

    PubMed Central

    Kasperzyk, Julie L.; Finn, Stephen P.; Flavin, Richard; Fiorentino, Michelangelo; Lis, Rosina; Hendrickson, Whitney K.; Clinton, Steven K.; Sesso, Howard D.; Giovannucci, Edward L.; Stampfer, Meir J.; Loda, Massimo; Mucci, Lorelei A.

    2013-01-01

    Background Over-expression of prostate-specific membrane antigen (PSMA) in tumor tissue and serum has been linked to increased risk of biochemical recurrence in surgically treated prostate cancer patients, but no studies have assessed its association with disease-specific mortality. Methods We examined whether high PSMA protein expression in prostate tumor tissue was associated with lethal disease, and with tumor biomarkers of progression, among participants of two US-based cohorts (n=902, diagnosed 1983–2004). We used Cox proportional hazards regression to calculate multivariable hazard ratios (HR) and 95% confidence intervals (CI) of lethal prostate cancer, defined as disease-specific death or development of distant metastases (n=95). Partial Spearman rank correlation coefficients were used to correlate PSMA with tumor biomarkers. Results During an average 13 years of follow-up, higher PSMA expression at prostatectomy was significantly associated with lethal prostate cancer (age-adjusted HRQuartile(Q)4vs.Q1=2.42; p-trend<0.01). This association was attenuated and non-significant (multivariable-adjusted HRQ4vs.Q1=1.01; p-trend=0.52) after further adjusting for Gleason score and PSA at diagnosis. High PSMA expression was significantly (p<0.05) correlated with higher Gleason score and PSA at diagnosis, increased tumor angiogenesis, lower vitamin D receptor and androgen receptor expression, and absence of ERG expression. Conclusions High tumor PSMA expression was not an independent predictor of lethal prostate cancer in the current study. PSMA expression likely captures, in part, malignant features of Gleason grade and tumor angiogenesis. Impact PSMA is not a strong candidate biomarker for predicting prostate cancer-specific mortality in surgically treated patients. PMID:24130224

  18. Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers

    PubMed Central

    Polisetty, Ravindra Varma; Gautam, Poonam; Gupta, Manoj Kumar; Sharma, Rakesh; Gowda, Harsha; Renu, Durairaj; Shivakumar, Bhadravathi Marigowda; Lakshmikantha, Akhila; Mariswamappa, Kiran; Ankathi, Praveen; Purohit, Aniruddh K.; Uppin, Megha S.; Sundaram, Challa; Sirdeshmukh, Ravi

    2016-01-01

    Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients. PMID:27246909

  19. Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers.

    PubMed

    Polisetty, Ravindra Varma; Gautam, Poonam; Gupta, Manoj Kumar; Sharma, Rakesh; Gowda, Harsha; Renu, Durairaj; Shivakumar, Bhadravathi Marigowda; Lakshmikantha, Akhila; Mariswamappa, Kiran; Ankathi, Praveen; Purohit, Aniruddh K; Uppin, Megha S; Sundaram, Challa; Sirdeshmukh, Ravi

    2016-01-01

    Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients. PMID:27246909

  20. Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

    PubMed

    Barandika, Olatz; Ezquerra-Inchausti, Maitane; Anasagasti, Ander; Vallejo-Illarramendi, Ainara; Llarena, Irantzu; Bascaran, Lucia; Alberdi, Txomin; De Benedetti, Giacomo; Mendicute, Javier; Ruiz-Ederra, Javier

    2016-10-01

    In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate. PMID:27497833

  1. Heterologous Expression and Purification Systems for Structural Proteomics of Mammalian Membrane Proteins

    PubMed Central

    2002-01-01

    Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural proteomics. PMID:18629259

  2. Organic micropollutants in aerobic and anaerobic membrane bioreactors: Changes in microbial communities and gene expression.

    PubMed

    Harb, Moustapha; Wei, Chun-Hai; Wang, Nan; Amy, Gary; Hong, Pei-Ying

    2016-10-01

    Organic micro-pollutants (OMPs) are contaminants of emerging concern in wastewater treatment due to the risk of their proliferation into the environment, but their impact on the biological treatment process is not well understood. The purpose of this study is to examine the effects of the presence of OMPs on the core microbial populations of wastewater treatment. Two nanofiltration-coupled membrane bioreactors (aerobic and anaerobic) were subjected to the same operating conditions while treating synthetic municipal wastewater spiked with OMPs. Microbial community dynamics, gene expression levels, and antibiotic resistance genes were analyzed using molecular-based approaches. Results showed that presence of OMPs in the wastewater feed had a clear effect on keystone bacterial populations in both the aerobic and anaerobic sludge while also significantly impacting biodegradation-associated gene expression levels. Finally, multiple antibiotic-type OMPs were found to have higher removal rates in the anaerobic MBR, while associated antibiotic resistance genes were lower. PMID:27441825

  3. Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays.

    PubMed

    Heinen, Robert B; Bienert, Gerd Patrick; Cohen, David; Chevalier, Adrien S; Uehlein, Norbert; Hachez, Charles; Kaldenhoff, Ralf; Le Thiec, Didier; Chaumont, François

    2014-10-01

    Stomata, the microscopic pores on the surface of the aerial parts of plants, are bordered by two specialized cells, known as guard cells, which control the stomatal aperture according to endogenous and environmental signals. Like most movements occurring in plants, the opening and closing of stomata are based on hydraulic forces. During opening, the activation of plasma membrane and tonoplast transporters results in solute accumulation in the guard cells. To re-establish the perturbed osmotic equilibrium, water follows the solutes into the cells, leading to their swelling. Numerous studies have contributed to the understanding of the mechanism and regulation of stomatal movements. However, despite the importance of transmembrane water flow during this process, only a few studies have provided evidence for the involvement of water channels, called aquaporins. Here, we microdissected Zea mays stomatal complexes and showed that members of the aquaporin plasma membrane intrinsic protein (PIP) subfamily are expressed in these complexes and that their mRNA expression generally follows a diurnal pattern. The substrate specificity of two of the expressed ZmPIPs, ZmPIP1;5 and ZmPIP1;6, was investigated by heterologous expression in Xenopus oocytes and yeast cells. Our data show that both isoforms facilitate transmembrane water diffusion in the presence of the ZmPIP2;1 isoform. In addition, both display CO2 permeability comparable to that of the CO2 diffusion facilitator NtAQP1. These data indicate that ZmPIPs may have various physiological roles in stomatal complexes. PMID:25082269

  4. Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation

    SciTech Connect

    Ji, Peng; Lodish, Harvey F.

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Ankyrin and band 3 are not required for erythroblasts enucleation. Black-Right-Pointing-Pointer Loss of ankyrin does not affect erythroid membrane glycoprotein expression. Black-Right-Pointing-Pointer Loss of band 3 influences erythroid membrane glycoprotein expression. -- Abstract: During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex.

  5. Cell-based capacitance sensor for analysis of EGFR expression on cell membrane

    NASA Astrophysics Data System (ADS)

    Shin, Dong-Myeong; Shin, Yong-Cheol; Ha, Ji Hye; Lee, Jong-Ho; Han, Dong-Wook; Kim, Jong-Man; Kim, Hyung Kook; Hwang, Yoon-Hwae

    2013-02-01

    Cancer cells have many kinds of cancer biomarkers. Among them, the epidermal growth factor (EGF) receptors can show a possibility for a cancer marker because the over-expression of EGF receptor is related with fibrous, colorectal, cervical and gastric tumorigenesis. We fabricated the capacitance sensor with a gap area of 50 μm × 200 μm by using photolithography and lift-off method. Using the capacitance sensor, we investigated the time dependent capacitance changes of different kinds of fibrous cells, such as HT1080 fibrosarcoma, L-929 fibroblast cell line and nHDF dermal fibroblast primary cell. We found that when we put the EGF, the capacitance decreased due to the immobilization of EGF to EGF receptor on the cell membrane. The quantitative determination of EGF receptor level for various fibrous cells was carried out and the results showed good correlation with conventional method. Based on our results, we suggest that the capacitance sensor can measure the expression level of the EGF receptor on cell membrane and be a good candidate as a cancer diagnosis.

  6. Potassium-dependent changes in the expression of membrane-associated proteins in barley roots

    SciTech Connect

    Fernando, M.; Kulpa, J.; Siddiqi, M.Y.; Glass, A.D.M. )

    1990-04-01

    Barley (Hordeum vulgare L. cv Halcyon) seedlings which has been grown in full strength complete inorganic nutrient media (containing 6 millimolar K{sup +}) had high internal K{sup +} concentrations and low values of K{sup +} ({sup 86}Rb{sup +}) influx when influx was measured from solutions containing 100 micromolar K{sup +}. Transfer of these plants to solutions lacking K{sup +} resulted in significant reductions of root and shoot K{sup +} concentrations and values of K{sup +} ({sup 86}Rb{sup +}) influx increased by greater than 10-fold within 3 days. When plants treated in this way were returned to complete solutions, containing K{sup +}, the changes induced by K{sup +} deprivation were reversed. Parallel studies of microsomal membranes by means of SDS-PAGE demonstrated that the expression of a group of polypeptides increased or decreased in parallel with changes of K{sup +} ({sup 86}Rb{sup +}) influx. Most prominent of these were 45 and 34 kilodalton polypeptides which specifically responded to K{sup +} status of the barley plants; their expression was not enhanced by N or P deprivation. The 45 kilodalton polypeptide was susceptible to degradation by a membrane associated protease when microsomes were washing in buffer containing 0.2 millimolar PMSF. This loss was prevented by increasing PMSF concentration to 2 millimolar.

  7. Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family.

    PubMed

    Guo, Lei; Wang, Zi Yi; Lin, Hong; Cui, Wei Er; Chen, Jun; Liu, Meihua; Chen, Zhang Liang; Qu, Li Jia; Gu, Hongya

    2006-03-01

    Plasma membrane intrinsic proteins (PIPs) are a subfamily of aquaporins that enable fast and controlled translocation of water across the membrane. In this study, we systematically identified and cloned ten PIP genes from rice. Based on the similarity of the amino acid sequences they encoded, these rice PIP genes were classified into two groups and designated as OsPIP1-1 to OsPIP1-3 and OsPIP2-1 to OsPIP2-7 following the nomenclature of PIP genes in maize. Quantitative RT-PCR analysis identified three root-specific and one leaf-specific OsPIP genes. Furthermore, the expression profile of each OsPIP gene in response to salt, drought and ABA treatment was examined in detail. Analysis on transgenic plants over-expressing of either OsPIP1 (OsPIP1-1) or OsPIP2 (OsPIP2-2) in wild-type Arabidopsis, showed enhanced tolerance to salt (100 mM of NaCl) and drought (200 mM of mannitol), but not to salt treatment of higher concentration (150 mM of NaCl). Taken together, these data suggest a distinct role of each OsPIP gene in response to different stresses, and should add a new layer to the understanding of the physiological function of rice PIP genes. PMID:16541126

  8. Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).

    PubMed Central

    Champion, C I; Blanco, D R; Exner, M M; Erdjument-Bromage, H; Hancock, R E; Tempst, P; Miller, J N; Lovett, M A

    1997-01-01

    In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum. PMID:9023206

  9. Effect of UV irradiation on expression of membrane IL 1 by rat macrophages

    SciTech Connect

    Lange-Wantzin, G.; Rothlein, R.; Kahn, J.; Faanes, R.B.

    1987-06-01

    The effect of UV-B irradiation on the expression of membrane-associated IL 1 (mIL 1) by rat pulmonary alveolar macrophages (PAM) was studied. We found that although there was an increase in secreted IL 1 by PAM exposed to UV-B, the expression of mIL 1 was inhibited in a dose-dependent manner. Furthermore, PAM that were allowed to express mIL 1 before UV-B irradiation had a faster decay of mIL 1 activity than unirradiated cells. These data suggested that mIL 1 expression is inhibited by UV-B irradiation, and that under normal circumstances, mIL 1 synthesis and degradation is at a steady state, with the half-life of mIL 1 activity being 24 hr when assayed in an IL 1-dependent cell line proliferation assay. These data indicate that secreted forms of IL 1 and mIL 1 are differentially regulated and that the therapeutic effects of UV irradiation may be due to its inhibition of mIL 1 activity.

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

    PubMed Central

    Santhanam, Abirami; Torricelli, Andre A. M.; Wu, Jiahui; Marino, Gustavo K.

    2015-01-01

    Purpose 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). Methods 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. Results 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

  11. Prognostic Significance of EBV Latent Membrane Protein 1 Expression in Lymphomas: Evidence from 15 Studies

    PubMed Central

    Mao, Yuan; Lu, Mei Ping; Lin, Hong; Zhang, Da Wei; Liu, Ying; Li, Qing Dong; Lv, Zhi Gang; Xu, Jia Ren; Chen, Ren Jie; Zhu, Jin

    2013-01-01

    Background Epstein-Barr virus (EBV) infection has been associated with lymphoma development. EBV latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation and progression of different human cells, including lymphocytes. This meta-analysis investigated LMP1 expression with prognosis of patients with lymphoma. Methods The electronic databases of PubMed, Embase, and Chinese Biomedicine Databases were searched. There were 15 published studies available for a random effects model analysis. Quality assessment was performed using the Newcastle-Ottawa Quality Assessment Scale for cohort studies. A funnel plot was used to investigate publication bias, and sources of heterogeneity were identified by meta-regression analysis. The combined hazard ratios (HR) and their corresponding 95% confidence intervals of LMP1 expression were calculated by comparison to the overall survival. Results Overall, there was no statistical significance found between LMP1 expression and survival of lymphoma patients (HR 1.25 [95% CI, 0.92–1.68]). In subgroup analyses, LMP1 expression was associated with survival in patients with non-Hodgkin lymphoma (NHL) (HR  = 1.84, 95% CI: 1.02–3.34), but not with survival of patients with Hodgkin disease (HD) (HR  =  1.03, 95% CI: 0.74–1.44). In addition, significant heterogeneity was present and the meta-regression revealed that the outcome of analysis was mainly influenced by the cutoff value. Conclusions This meta-analysis demonstrated that LMP1 expression appears to be an unfavorable prognostic factor for overall survival of NHL patients. The data suggested that EBV infection and LMP1 expression may be an important factor for NHL development or progression. PMID:23613723

  12. Intraepidermal expression of basement membrane components in the lesional skin of a patient with dystrophic epidermolysis bullosa.

    PubMed

    Muramatsu, T; Ko, T; Honoki, K; Hatoko, M; Shirai, T; Vnittanakom, P

    1999-02-01

    The patient was a 15-year-old male. Since birth, he had developed blistering and erosion of the skin. Biopsy skin specimen of the bullous lesions showed subepidermal blister formation. Electron microscopic examination revealed that tissue separation had occurred at the sublamina densa level. By indirect immunofluorescence using antibodies specific for alpha 6 integrin, laminin 5, type IV collagen, and type VII collagen, all of these basement membrane components were detected as coarse granular intracytoplasmic deposits only in the basal and suprabasal cells of the blister roof. In the non-blistered regions, these basement membrane components showed a linear pattern similar to that seen in normal skin. These findings suggest that intraepidermal expression of basement membrane components was closely related to the blister formation. The biological meaning of intraepidermal expression of basement membrane components were also discussed. PMID:10091480

  13. Proteomic Analysis of the Rat Canalicular Membrane Reveals Expression of a Complex System of P4-ATPases in Liver

    PubMed Central

    Chaubey, Pururawa Mayank; Hofstetter, Lia; Roschitzki, Bernd; Stieger, Bruno

    2016-01-01

    Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes. PMID:27347675

  14. Proteomic Analysis of the Rat Canalicular Membrane Reveals Expression of a Complex System of P4-ATPases in Liver.

    PubMed

    Chaubey, Pururawa Mayank; Hofstetter, Lia; Roschitzki, Bernd; Stieger, Bruno

    2016-01-01

    Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes. PMID:27347675

  15. Gene expression analysis of membrane transporters and drug-metabolizing enzymes in the lung of healthy and COPD subjects

    PubMed Central

    Berg, Tove; Hegelund Myrbäck, Tove; Olsson, Marita; Seidegård, Janeric; Werkström, Viktoria; Zhou, Xiao-Hong; Grunewald, Johan; Gustavsson, Lena; Nord, Magnus

    2014-01-01

    This study describes for the first time the expression levels of genes encoding membrane transporters and drug-metabolizing enzymes in the lungs of ex-smoking patients with chronic obstructive pulmonary disease (COPD). Membrane transporters and drug-metabolizing enzymes are key determinants of drug uptake, metabolism, and elimination for systemically administered as well as inhaled drugs, with consequent influence on clinical efficacy and patient safety. In this study, while no difference in gene expression was found between healthy and COPD subjects, we identified a significant regional difference in mRNA expression of both membrane transporters and drug-metabolizing enzymes between central and peripheral tissue in both healthy and COPD subjects. The majority of the differentially expressed genes were higher expressed in the central airways such as the transporters SLC2A1 (GLUT1), SLC28A3 (CNT3), and SLC22A4 (OCTN1) and the drug-metabolizing enzymes GSTZ1, GSTO2, and CYP2F1. Together, this increased knowledge of local pharmacokinetics in diseased and normal lung may improve modeling of clinical outcomes of new chemical entities intended for inhalation therapy delivered to COPD patients. In addition, based on the similarities between COPD and healthy subjects regarding gene expression of membrane transporters and drug-metabolizing enzymes, our results suggest that clinical pharmacological studies in healthy volunteers could be a valid model of COPD patients regarding drug disposition of inhaled drugs in terms of drug metabolism and drug transporters. PMID:25505599

  16. Expression and Functions of CreD, an Inner Membrane Protein in Stenotrophomonas maltophilia

    PubMed Central

    Huang, Hsin-Hui; Lin, Yi-Tsung; Chen, Wei-Ching; Huang, Yi-Wei; Chen, Shiang-Jiuun; Yang, Tsuey-Ching

    2015-01-01

    CreBC is a highly conserved two-component regulatory system (TCS) in several gram-negative bacteria, including Escherichia coli, Aeromonas spp., Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. CreD is a conserved gene that encodes a predicted inner-membrane protein and is located near the creBC loci. Activation of CreBC increases creD expression; therefore, creD expression is generally used as a measure of CreBC activation in E. coli, Aeromonas spp., and P. aeruginosa systems. In this article, we aim to elucidate the expression of creD and further to investigate its functions in S. maltophilia. In spite of a short intergenic region of 81 bp between creBC and creD, creD is expressed separately from the adjacent creBC operon and from a promoter immediately upstream of creD (PcreD) in S. maltophilia. We found that the promoter activity of PcreD is negatively regulated by the creBC TCS, positively regulated by the bacterial culture density, and not affected by β-lactams. Furthermore, creD expression is not significantly altered in the presence of the phosphor-mimic variant of CreB, CreB(D55E), which mimics activated CreB. The functions of CreD of S. maltophilia were assessed by comparison among the following: wild-type KJ; the creD isogenic mutant, KJΔCreD; and the complementary strain, KJΔCreD(pCreD). The mutant lacking creD had cell division defects and aberrations in cell envelope integrity, which then triggered the σE-mediated envelope stress response. Thus, the results indicated that CreD plays a critical role in the maintenance of envelope integrity. PMID:26698119

  17. Expression of the major outer membrane protein of Chlamydia trachomatis in Escherichia coli.

    PubMed Central

    Manning, D S; Stewart, S J

    1993-01-01

    The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein. Images PMID:8406797

  18. Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa.

    PubMed

    Arduino, Paolo G; Carrozzo, Marco; Pagano, Marco; Broccoletti, Roberto; Scully, Crispian; Gandolfo, Sergio

    2010-06-01

    Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone. PMID:19506920

  19. Light-harvesting processes in the dynamic photosynthetic antenna.

    PubMed

    Duffy, C D P; Valkunas, L; Ruban, A V

    2013-11-21

    We present our perspective on the theoretical basis of light-harvesting within the photosynthetic membrane. Far from being a static structure, the photosynthetic membrane is a highly dynamic system, with protein mobility playing an important role in the damage/repair cycle of photosystem II (PSII), in balancing the input of energy between PSI and PSII, and in the photoprotection of PSII in response to a sudden excess of illumination. The concept of a photosynthetic antenna is illustrated and the state transition phenomenon is discussed as an example of purposeful antenna mobility. We discuss fluorescence recovery after photo-bleaching as a technique for visualising membrane mobility, before introducing light-induced grana membrane reorganisation as an integral part of the rapid photoprotective switch in plants. We then discuss current theoretical approaches to modelling the energy transfer dynamics of the PSII antenna: the atomistic models of intra-complex transfer and the coarse-grained approach to the inter-complex dynamics. Finally we discuss the future prospect of extending these methods, beyond the static picture of the membrane, to the dynamic PSII photosynthetic antenna. PMID:23868502

  20. Low plasma membrane expression of the miltefosine transport complex renders Leishmania braziliensis refractory to the drug.

    PubMed

    Sánchez-Cañete, María P; Carvalho, Luís; Pérez-Victoria, F Javier; Gamarro, Francisco; Castanys, Santiago

    2009-04-01

    Miltefosine (hexadecylphosphocholine, MLF) is the first oral drug with recognized efficacy against both visceral and cutaneous leishmaniasis. However, some clinical studies have suggested that MLF shows significantly less efficiency against the cutaneous leishmaniasis caused by Leishmania braziliensis. In this work, we have determined the cellular and molecular basis for the natural MLF resistance observed in L. braziliensis. Four independent L. braziliensis clinical isolates showed a marked decrease in MLF sensitivity that was due to their inability to internalize the drug. MLF internalization in the highly sensitive L. donovani species requires at least two proteins in the plasma membrane, LdMT, a P-type ATPase involved in phospholipid translocation, and its beta subunit, LdRos3. Strikingly, L. braziliensis parasites showed highly reduced levels of this MLF translocation machinery at the plasma membrane, mainly because of the low expression levels of the beta subunit, LbRos3. Overexpression of LbRos3 induces increased MLF sensitivity not only in L. braziliensis promastigotes but also in intracellular amastigotes. These results further highlight the importance of the MLF translocation machinery in determining MLF potency and point toward the development of protocols to routinely monitor MLF susceptibility in geographic areas where L. braziliensis might be prevalent. PMID:19188379

  1. The role of putrescine in the regulation of proteins and fatty acids of thylakoid membranes under salt stress.

    PubMed

    Shu, Sheng; Yuan, Yinghui; Chen, Jie; Sun, Jin; Zhang, Wenhua; Tang, Yuanyuan; Zhong, Min; Guo, Shirong

    2015-01-01

    Polyamines can alleviate the inhibitory effects of salinity on plant growth by regulating photosynthetic efficiency. However, little information is available to explain the specific mechanisms underlying the contribution of polyamines to salt tolerance of the photosynthetic apparatus. Here, we investigated the role of putrescine (Put) on the photosynthetic apparatus of cucumber seedlings under salt stress. We found that NaCl stress resulted in severe ion toxicity and oxidative stress in cucumber chloroplasts. In addition, salinity caused a significant increase in the saturated fatty acid contents of thylakoid membranes. Put altered unsaturated fatty acid content, thereby alleviating the disintegration of thylakoid grana lamellae and reducing the number of plastoglobuli in thylakoid membranes. BN-PAGE revealed Put up-regulated the expression of ATP synthase, CP47, D1, Qb, and psbA proteins and down-regulated CP24, D2, and LHCII type III in NaCl-stressed thylakoid membranes. qRT-PCR analysis of gene expression was used to compare transcript and protein accumulation among 10 candidate proteins. For five of these proteins, induced transcript accumulation was consistent with the pattern of induced protein accumulation. Our results suggest that Put regulates protein expression at transcriptional and translational levels by increasing endogenous polyamines levels in thylakoid membranes, which may stabilise photosynthetic apparatus under salt stress. PMID:26435404

  2. The role of putrescine in the regulation of proteins and fatty acids of thylakoid membranes under salt stress

    PubMed Central

    Shu, Sheng; Yuan, Yinghui; Chen, Jie; Sun, Jin; Zhang, Wenhua; Tang, Yuanyuan; Zhong, Min; Guo, Shirong

    2015-01-01

    Polyamines can alleviate the inhibitory effects of salinity on plant growth by regulating photosynthetic efficiency. However, little information is available to explain the specific mechanisms underlying the contribution of polyamines to salt tolerance of the photosynthetic apparatus. Here, we investigated the role of putrescine (Put) on the photosynthetic apparatus of cucumber seedlings under salt stress. We found that NaCl stress resulted in severe ion toxicity and oxidative stress in cucumber chloroplasts. In addition, salinity caused a significant increase in the saturated fatty acid contents of thylakoid membranes. Put altered unsaturated fatty acid content, thereby alleviating the disintegration of thylakoid grana lamellae and reducing the number of plastoglobuli in thylakoid membranes. BN-PAGE revealed Put up-regulated the expression of ATP synthase, CP47, D1, Qb, and psbA proteins and down-regulated CP24, D2, and LHCII type III in NaCl-stressed thylakoid membranes. qRT-PCR analysis of gene expression was used to compare transcript and protein accumulation among 10 candidate proteins. For five of these proteins, induced transcript accumulation was consistent with the pattern of induced protein accumulation. Our results suggest that Put regulates protein expression at transcriptional and translational levels by increasing endogenous polyamines levels in thylakoid membranes, which may stabilise photosynthetic apparatus under salt stress. PMID:26435404

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  5. High-Yield Expression of a Catalytically Active Membrane-Bound Protein: Human P450 Oxidoreductase

    PubMed Central

    Sandee, Duanpen

    2011-01-01

    P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 μm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 μm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins. PMID:21586563

  6. Membrane Systems in Cyanobacteria

    SciTech Connect

    Liberton, Michelle L.; Pakrasi, Himadri B.

    2008-01-01

    Cyanobacteria are photosynthetic prokaryotes with highly differentiated membrane systems. In addition to a Gram-negative-type cell envelope with plasma membrane and outer membrane separated by a periplasmic space, cyanobacteria have an internal system of thylakoid membranes where the fully functional electron transfer chains of photosynthesis and respiration reside. The presence of different membrane systems lends these cells a unique complexity among bacteria. Cyanobacteria must be able to reorganize the membranes, synthesize new membrane lipids, and properly target proteins to the correct membrane system. The outer membrane, plasma membrane, and thylakoid membranes each have specialized roles in the cyanobacterial cell. Understanding the organization, functionality, protein composition and dynamics of the membrane systems remains a great challenge in cyanobacterial cell biology.

  7. Efficiency of Membrane Protein Expression Following Infection with Recombinant Adenovirus of Polarized Non-Transformed Human Retinal Pigment Epithelial Cells.

    PubMed

    Müller, Claudia; Blenkinsop, Timothy A; Stern, Jeffrey H; Finnemann, Silvia C

    2016-01-01

    Transient expression of exogenous proteins facilitates studies of molecular mechanisms and utility for transplantation of retinal pigment epithelial (RPE) cells in culture. Here, we compared expression of the membrane protein β5 integrin-GFP (β5-GFP) in two recently established models of differentiated human RPE, adult RPE stem cell-derived RPE and primary fetal RPE, upon infection with recombinant adenovirus or transfection with DNA in liposomes. We varied viral titer and duration of virus incubation and examined β5-GFP and the tight junction marker ZO-1 in manipulated cells by confocal microscopy. Fewer than 5 % of cells expressed β5-GFP after liposome-mediated transfection. The percentage of cells with detectable β5-GFP exceeded 90 % after adenovirus infection for as little as 1 h. Decreasing virus titer two-fold did not alter the fraction of cells expressing β5-GFP but increased variability of β5-GFP level among cells. In cells with low expression levels, β5-GFP localized mostly to the apical plasma membrane like endogenous αvβ5 integrin. In cells with high expression levels, β5-GFP localized to the cytoplasm in addition to the apical surface suggesting accumulation in trafficking compartments. Altogether, adenovirus delivery yields efficient exogenous membrane protein expression of correct polarity in differentiated human RPE cells in culture. PMID:26427482

  8. Linking Gene Expression and Membrane Lipid Composition of Arabidopsis[W][OPEN

    PubMed Central

    Szymanski, Jedrzej; Brotman, Yariv; Willmitzer, Lothar; Cuadros-Inostroza, Álvaro

    2014-01-01

    Glycerolipid metabolism of plants responds dynamically to changes in light intensity and temperature, leading to the modification of membrane lipid composition to ensure optimal biochemical and physical properties in the new environment. Although multiple posttranscriptional regulatory mechanisms have been reported to be involved in the process, the contribution of transcriptional regulation remains largely unknown. Here, we present an integrative analysis of transcriptomic and lipidomic data, revealing large-scale coordination between gene expression and changes in glycerolipid levels during the Arabidopsis thaliana response to light and temperature stimuli. Using a multivariate regression technique called O2PLS, we show that the gene expression response is strictly coordinated at the biochemical pathway level and occurs in parallel with changes of specific glycerolipid pools. Five interesting candidate genes were chosen for further analysis from a larger set of candidates identified based on their close association with various groups of glycerolipids. Lipidomic analysis of knockout mutant lines of these five genes showed a significant relationship between the coordination of transcripts and glycerolipid levels in a changing environment and the effects of single gene perturbations. PMID:24642935

  9. Induced expression of neuronal membrane attack complex and cell death by Alzheimer's beta-amyloid peptide.

    PubMed

    Shen, Y; Sullivan, T; Lee, C M; Meri, S; Shiosaki, K; Lin, C W

    1998-06-15

    beta-amyloid peptide (A beta) and complement-derived membrane attack complex (MAC) are co-localized in senile plaques of brains from Alzheimer's disease (AD) patients. But the relationship between A beta and complement activation is unclear. We have used human neurotypic cells, differentiated SH-SY5Y, as a model system to examine regulation of neuronal MAC expression and cell death by A beta. We demonstrated that mRNAs (C1q, C2, C3, C4, C5, C6, C7, C8 and C9) and proteins (C1q, C3 and C9) for the major components of the classical complement cascade are present in the SH-SY5Y neurotypic cells, indicating that neuronal cells can synthesize the necessary proteins required for MAC formation. Furthermore, immunocytochemical studies showed the A beta-induced neuronal MAC expression on the SH-SY5Y cells after CD59 was removed by PIPLC or blocked by anti-CD59 antibody. Meanwhile, increased A beta-induced neuronal cell death was observed following treatment with anti-CD59. Taken together, these results suggest that A beta activates neuronal complement cascade to induce MAC, and a deficiency of endogenous complement regulatory proteins, e.g., CD59, may increase the vulnerability of neurons to complement-mediated cytotoxicity. PMID:9689469

  10. Theory of fluorescence induction in photosystem II: derivation of analytical expressions in a model including exciton-radical-pair equilibrium and restricted energy transfer between photosynthetic units.

    PubMed Central

    Lavergne, J; Trissl, H W

    1995-01-01

    The theoretical relationships between the fluorescence and photochemical yields of PS II and the fraction of open reaction centers are examined in a general model endowed with the following features: i) a homogeneous, infinite PS II domain; ii) exciton-radical-pair equilibrium; and iii) different rates of exciton transfer between core and peripheral antenna beds. Simple analytical relations are derived for the yields and their time courses in induction experiments. The introduction of the exciton-radical-pair equilibrium, for both the open and closed states of the trap, is shown to be equivalent to an irreversible trapping scheme with modified parameters. Variation of the interunit transfer rate allows continuous modulation from the case of separated units to the pure lake model. Broadly used relations for estimating the relative amount of reaction centers from the complementary area of the fluorescence kinetics or the photochemical yield from fluorescence levels are examined in this framework. Their dependence on parameters controlling exciton decay is discussed, allowing assessment of their range of applicability. An experimental induction curve is analyzed, with a discussion of its decomposition into alpha and beta contributions. The sigmoidicity of the induction kinetics is characterized by a single parameter J related to Joliot's p, which is shown to depend on both the connectivity of the photosynthetic units and reaction center parameters. On the other hand, the relation between J and the extreme fluorescence levels (or the deviation from the linear Stern-Volmer dependence of 1/phi f on the fraction of open traps) is controlled only by antenna connectivity. Experimental data are consistent with a model of connected units for PS II alpha, intermediate between the pure lake model of unrestricted exciton transfer and the isolated units model. PMID:7647250

  11. Statin Therapy and the Expression of Genes that Regulate Calcium Homeostasis and Membrane Repair in Skeletal Muscle

    PubMed Central

    Draeger, Annette; Sanchez-Freire, Verónica; Monastyrskaya, Katia; Hoppeler, Hans; Mueller, Matthias; Breil, Fabio; Mohaupt, Markus G.; Babiychuk, Eduard B.

    2010-01-01

    In skeletal muscle of patients with clinically diagnosed statin-associated myopathy, discrete signs of structural damage predominantly localize to the T-tubular region and are suggestive of a calcium leak. The impact of statins on skeletal muscle of non-myopathic patients is not known. We analyzed the expression of selected genes implicated in the molecular regulation of calcium and membrane repair, in lipid homeostasis, myocyte remodeling and mitochondrial function. Microscopic and gene expression analyses were performed using validated TaqMan custom arrays on skeletal muscle biopsies of 72 age-matched subjects who were receiving statin therapy (n = 38), who had discontinued therapy due to statin-associated myopathy (n = 14), and who had never undergone statin treatment (n = 20). In skeletal muscle, obtained from statin-treated, non-myopathic patients, statins caused extensive changes in the expression of genes of the calcium regulatory and the membrane repair machinery, whereas the expression of genes responsible for mitochondrial function or myocyte remodeling was unaffected. Discontinuation of treatment due to myopathic symptoms led to a normalization of gene expression levels, the genes encoding the ryanodine receptor 3, calpain 3, and dystrophin being the most notable exceptions. Hence, even in clinically asymptomatic (non-myopathic) patients, statin therapy leads to an upregulation in the expression of genes that are concerned with skeletal muscle regulation and membrane repair. PMID:20489141

  12. Interleukin-1-induced gene expression requires the membrane-raft-dependent internalization of the interleukin-1 receptor.

    PubMed

    Windheim, Mark

    2016-10-01

    Interleukin-1 (IL-1) binding to its receptor triggers signaling events at the plasma membrane that are essential but not sufficient for the induction of the IL-1-dependent gene expression. In addition, the ligand-induced endocytosis of the IL-1 receptor and signaling events that are initiated after the internalization of the IL-1 receptor presumably involving signaling endosomes are critical for the IL-1-induced gene expression. In this study, we investigate the role of membrane domains, commonly denoted as lipid rafts, in the IL-1-induced signal transduction. We demonstrate that the internalization of the IL-1 receptor depends on the integrity of lipid rafts and that the disruption of lipid rafts strongly reduces the IL-1-induced gene expression. Interestingly, the IL-1-dependent signaling events activated at the plasma membrane are not influenced by the disruption of lipid rafts suggesting that IL-1 signaling is initiated in a non-raft domain of the plasma membrane. Subsequently, the IL-1 receptor is translocated to lipid rafts where receptor endocytosis occurs to enable the internalization-dependent IL-1 signaling to activate the IL-1-induced gene expression. PMID:27327966

  13. Channel Catfish, Ictalurus punctatus Rafinesque 1818, Tetraspanin Membrane Protein Family: Characterization and Expression Analysis of CD81 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CD81, also known as the target of an antiproliferative antibody 1 (TAPA-1), is a member of tetraspanin integral membrane protein family. This protein plays many important roles in immune functions. In this report, we characterized and analyzed expression of the channel catfish CD81 transcript. T...

  14. Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae

    PubMed Central

    Buck, Teresa M.; Jordan, Rick; Lyons-Weiler, James; Adelman, Joshua L.; Needham, Patrick G.; Kleyman, Thomas R.

    2015-01-01

    Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses. PMID:25759377

  15. Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from Mycobacterium avium subsp. paratuberculosis Strains K-10 and 187.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known of the protein expression in Mycobacterium avium subspecies paratuberculosis (MAP) and how this contributes to pathogenesis. In the present study, proteins from both outer membranes and cytosol were prepared from two strains of MAP; a laboratory-adapted strain K-10 and a recent isola...

  16. Alterations in cortisol concentrations and expression of certain genes associated with neutrophil functions in cows developing retention of fetal membranes.

    PubMed

    Pathak, Rupal; Prasad, Shiv; Kumaresan, A; Kaur, M; Manimaran, A; Dang, A K

    2015-12-15

    Elevated cortisol concentrations have been reported to impair the functions and alter the life span of neutrophils in cows. The present study assessed the cortisol concentrations and expression of few genes related to longevity (Fas, Caspase 3, Bcl2) and margination (CD 62L, CD 18/11b) of neutrophils in relation to retention of fetal membranes (RFM) in dairy cows. Cortisol concentrations were significantly higher on the day of calving and day 2 postpartum in RFM cows than normal cows. Expression of CD 62L was significantly lower on the day of calving and day 2 postpartum in RFM cows than normal cows. While expression of Fas and GR was significantly lower on the day of calving, expression of Bcl2 was lower on day 7±2 pre-partum in RFM cows compared to normal cows. No significant difference was observed in the expression of CD 18/11b and Caspase 3 between RFM and normal cows. Cortisol concentration was negatively correlated with expression of GR, Fas, CD 62L, CD18/CD11b and Caspase 3, while positively correlated with immature neutrophil percentage and expression of Bcl2. It may be inferred that cortisol concentrations and expression of certain genes associated with lifespan and margination of neutrophils were altered in cows developing RFM compared to those expelled the fetal membranes normally. PMID:26384698

  17. Regulation of the photosynthetic apparatus under fluctuating growth light.

    PubMed

    Tikkanen, Mikko; Grieco, Michele; Nurmi, Markus; Rantala, Marjaana; Suorsa, Marjaana; Aro, Eva-Mari

    2012-12-19

    Safe and efficient conversion of solar energy to metabolic energy by plants is based on tightly inter-regulated transfer of excitation energy, electrons and protons in the photosynthetic machinery according to the availability of light energy, as well as the needs and restrictions of metabolism itself. Plants have mechanisms to enhance the capture of energy when light is limited for growth and development. Also, when energy is in excess, the photosynthetic machinery slows down the electron transfer reactions in order to prevent the production of reactive oxygen species and the consequent damage of the photosynthetic machinery. In this opinion paper, we present a partially hypothetical scheme describing how the photosynthetic machinery controls the flow of energy and electrons in order to enable the maintenance of photosynthetic activity in nature under continual fluctuations in white light intensity. We discuss the roles of light-harvesting II protein phosphorylation, thermal dissipation of excess energy and the control of electron transfer by cytochrome b(6)f, and the role of dynamically regulated turnover of photosystem II in the maintenance of the photosynthetic machinery. We present a new hypothesis suggesting that most of the regulation in the thylakoid membrane occurs in order to prevent oxidative damage of photosystem I. PMID:23148275

  18. Regulation of the photosynthetic apparatus under fluctuating growth light

    PubMed Central

    Tikkanen, Mikko; Grieco, Michele; Nurmi, Markus; Rantala, Marjaana; Suorsa, Marjaana; Aro, Eva-Mari

    2012-01-01

    Safe and efficient conversion of solar energy to metabolic energy by plants is based on tightly inter-regulated transfer of excitation energy, electrons and protons in the photosynthetic machinery according to the availability of light energy, as well as the needs and restrictions of metabolism itself. Plants have mechanisms to enhance the capture of energy when light is limited for growth and development. Also, when energy is in excess, the photosynthetic machinery slows down the electron transfer reactions in order to prevent the production of reactive oxygen species and the consequent damage of the photosynthetic machinery. In this opinion paper, we present a partially hypothetical scheme describing how the photosynthetic machinery controls the flow of energy and electrons in order to enable the maintenance of photosynthetic activity in nature under continual fluctuations in white light intensity. We discuss the roles of light-harvesting II protein phosphorylation, thermal dissipation of excess energy and the control of electron transfer by cytochrome b6f, and the role of dynamically regulated turnover of photosystem II in the maintenance of the photosynthetic machinery. We present a new hypothesis suggesting that most of the regulation in the thylakoid membrane occurs in order to prevent oxidative damage of photosystem I. PMID:23148275

  19. A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status.

    PubMed

    Fusil, Floriane; Calattini, Sara; Amirache, Fouzia; Mancip, Jimmy; Costa, Caroline; Robbins, Justin B; Douam, Florian; Lavillette, Dimitri; Law, Mansun; Defrance, Thierry; Verhoeyen, Els; Cosset, François-Loïc

    2015-11-01

    The development of lentiviral vectors (LVs) for expression of a specific antibody can be achieved through the transduction of mature B-cells. This approach would provide a versatile tool for active immunotherapy strategies for infectious diseases or cancer, as well as for protein engineering. Here, we created a lentiviral expression system mimicking the natural production of these two distinct immunoglobulin isoforms. We designed a LV (FAM2-LV) expressing an anti-HCV-E2 surface glycoprotein antibody (AR3A) as a membrane-anchored Ig form or a soluble Ig form, depending on the B-cell maturation status. FAM2-LV induced high-level and functional membrane expression of the transgenic antibody in a nonsecretory B-cell line. In contrast, a plasma cell (PC) line transduced with FAM2-LV preferentially produced the secreted transgenic antibody. Similar results were obtained with primary B-cells transduced ex vivo. Most importantly, FAM2-LV transduced primary B-cells efficiently differentiated into PCs, which secreted the neutralizing anti-HCV E2 antibody upon adoptive transfer into immunodeficient NSG (NOD/SCIDγc(-/-)) recipient mice. Altogether, these results demonstrate that the conditional FAM2-LV allows preferential expression of the membrane-anchored form of an antiviral neutralizing antibody in B-cells and permits secretion of a soluble antibody following B-cell maturation into PCs in vivo. PMID:26281898

  20. Cold acclimation and BnCBF17-over-expression enhance photosynthetic performance and energy conversion efficiency during long-term growth of Brassica napus under elevated CO2 conditions.

    PubMed

    Dahal, Keshav; Gadapati, Winona; Savitch, Leonid V; Singh, Jas; Hüner, Norman P A

    2012-11-01

    The effects of cold acclimation and long-term elevated CO(2) on photosynthetic performance of wild-type (WT) and BnCBF17-over-expressing line of Brassica napus cv. Westar (BnCBF17-OE) grown at either 20/16 °C (non-acclimated) or 5/5 °C (cold acclimated) and at either ambient (380 μmol C mol(-1)) or elevated (700 μmol C mol(-1)) CO(2) were studied. Compared with non-acclimated WT, the BnCBF17-OE grown at 20 °C mimicked the effects of cold acclimation on WT B. napus with respect to compact dwarf phenotype and increased rates of light-saturated CO(2) assimilation and photosynthetic electron transport. This was associated with enhanced energy conversion efficiency into biomass as assessed by decreased excitation pressure coupled to decreased dependence on non-photochemical energy dissipation for a given irradiance. Growth at elevated CO(2) decreased the light and CO(2)-saturated rates of photosynthesis by 30 % for non-acclimated WT relative to growth at ambient CO(2). This was associated with inhibition in electron transport rates (20 %), decrease in amount of rbcL (35 %) and cytosolic FBPase (70 %) and increased excitation pressure and non-photochemical quenching in elevated versus ambient CO(2)-grown non-acclimated WT. In contrast, light and CO(2)-saturated rates of photosynthesis, electron transport, excitation pressure, non-photochemical quenching and levels of rbcL, cytosolic FBPase and Lhcb1 were insensitive to growth under elevated CO(2) in BnCBF17-OE and cold-acclimated WT. Thus, BnCBF17-over-expression and cold acclimation maintain enhanced energy conversion efficiency and reduced sensitivity to feedback-limited photosynthesis during long-term growth of B. napus under elevated CO(2). Our results indicated that CBFs transcription factors regulate not only freezing tolerance but also has major whole plant effects. PMID:22847022

  1. Loss of membranous VEGFR1 expression is associated with an adverse phenotype and shortened survival in breast cancer

    PubMed Central

    Lebok, Patrick; Huber, Julia; Burandt, Eike-Christian; Lebeau, Annette; Marx, Andreas Holger; Terracciano, Luigi; Heilenkötter, Uwe; Jänicke, Fritz; Müller, Volkmar; Paluchowski, Peter; Geist, Stefan; Wilke, Christian; Simon, Ronald; Sauter, Guido; Quaas, Alexander

    2016-01-01

    Angiogenesis is a key process in tumor growth and progression, which is controlled by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). In order to better understand the prevalence and prognostic value of VEGFR1 expression in breast cancer, a tissue microarray containing >2,100 breast cancer specimens, with clinical follow-up data, was analyzed by immunohistochemistry using an antibody directed against the membrane-bound full-length receptor protein. The results demonstrated that membranous VEGFR1 staining was detected in all (5 of 5) normal breast specimens. In carcinoma specimens, membranous staining was negative in 3.1%, weak in 6.3%, moderate in 10.9%, and strong in 79.7% of the 1,630 interpretable tissues. Strong staining was significantly associated with estrogen receptor and progesterone receptor expression, but was inversely associated with advanced tumor stage (P=0.0431), high Bloom-Richardson-Ellis Score for Breast Cancer grade and low Ki67 labeling index (both P<0.0001). Cancers with moderate to strong (high) VEGFR1 expression were associated with significantly improved overall survival, as compared with tumors exhibiting negative or weak (low) expression (P=0.0015). This association was also detected in the subset of nodal-positive cancers (P=0.0018), and in the subset of 185 patients who had received tamoxifen as the sole therapy (P=0.001). In conclusion, these data indicated that membrane-bound VEGFR1 is frequently expressed in normal and cancerous breast epithelium. In addition, reduced or lost VEGFR1 expression may serve as a marker for poor prognosis in patients with breast cancer, who might not optimally benefit from endocrine therapy. PMID:27357606

  2. Lessons from an α-Helical Membrane Enzyme: Expression, Purification, and Detergent Optimization for Biophysical and Structural Characterization.

    PubMed

    Johnson, Jennifer L; Kalyoncu, Sibel; Lieberman, Raquel L

    2016-01-01

    This chapter outlines the protocol developed in our lab to produce a multipass α-helical membrane protein. We present our work flow, from ortholog selection to protein purification, including molecular biology for plasmid construction, protein expression in E. coli, membrane isolation and detergent solubilization, protein purification and tag removal, biophysical assessment of protein stability in different detergents, and detergent concentration determination using thin-layer chromatography. We focus on results from our ongoing work with intramembrane aspartyl proteases from archaeal organisms. PMID:27485343

  3. Analysis of Shewanella oneidensis Membrane Protein Expression in Response to Electron Acceptor Availability

    SciTech Connect

    Giometti, Carol S.; Khare, Tripti; Verberkmoes, Nathan; O'Loughlin, Ed; Lindberg, Carl; Thompson, Melissa; Hettich, Robert

    2006-04-05

    Shewanella oneidensis MR-1, a gram negative metal-reducing bacterium, can utilize a large number of electron acceptors. In the natural environment, S. oneidensis utilizes insoluble metal oxides as well as soluble terminal electron acceptors. The purpose of this ERSP project is to identify differentially expressed proteins associated with the membranes of S. oneidensis MR-1 cells grown with different electron acceptors, including insoluble metal oxides. We hypothesize that through the use of surface labeling, subcellular fractionation, and a combination of proteome analysis tools, proteins involved in the reduction of different terminal electron acceptors will be elucidated. We are comparing the protein profiles from cells grown with the soluble electron acceptors oxygen and fumarate and with those from cells grown with the insoluble iron oxides goethite, ferrihydrite and lepidocrocite. Comparison of the cell surface proteins isolated from cells grown with oxygen or anaerobically with fumarate revealed an increase in the abundance of over 25 proteins in anaerobic cells, including agglutination protein and flagellin proteins along with the several hypothetical proteins. In addition, the surface protein composition of cells grown with the insoluble iron oxides varies considerably from the protein composition observed with either soluble electron acceptor as well as between the different insoluble acceptors.

  4. Selection for mutants altered in the expression or export of outer membrane porin OmpF.

    PubMed Central

    Sodergren, E J; Davidson, J; Taylor, R K; Silhavy, T J

    1985-01-01

    Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins. Images PMID:2987179

  5. Isoform-Specific Up-Regulation of Plasma Membrane Ca2+ATPase Expression During Colon and Gastric Cancer Cell Differentiation

    PubMed Central

    Ribiczey, Polett; Tordai, Attila; Andrikovics, Hajnalka; Filoteo, Adelaida G.; Penniston, John T.; Enouf, Jocelyne; Enyedi, Ágnes; Papp, Béla; Kovács, Tünde

    2007-01-01

    Summary In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype. PMID:17433436

  6. Towards autotrophic tissue engineering: Photosynthetic gene therapy for regeneration.

    PubMed

    Chávez, Myra Noemi; Schenck, Thilo Ludwig; Hopfner, Ursula; Centeno-Cerdas, Carolina; Somlai-Schweiger, Ian; Schwarz, Christian; Machens, Hans-Günther; Heikenwalder, Mathias; Bono, María Rosa; Allende, Miguel L; Nickelsen, Jörg; Egaña, José Tomás

    2016-01-01

    The use of artificial tissues in regenerative medicine is limited due to hypoxia. As a strategy to overcome this drawback, we have shown that photosynthetic biomaterials can produce and provide oxygen independently of blood perfusion by generating chimeric animal-plant tissues during dermal regeneration. In this work, we demonstrate the safety and efficacy of photosynthetic biomaterials in vivo after engraftment in a fully immunocompetent mouse skin defect model. Further, we show that it is also possible to genetically engineer such photosynthetic scaffolds to deliver other key molecules in addition to oxygen. As a proof-of-concept, biomaterials were loaded with gene modified microalgae expressing the angiogenic recombinant protein VEGF. Survival of the algae, growth factor delivery and regenerative potential were evaluated in vitro and in vivo. This work proposes the use of photosynthetic gene therapy in regenerative medicine and provides scientific evidence for the use of engineered microalgae as an alternative to deliver recombinant molecules for gene therapy. PMID:26474040

  7. Photosynthetic Pigments in Diatoms.

    PubMed

    Kuczynska, Paulina; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

    2015-09-01

    Photosynthetic pigments are bioactive compounds of great importance for the food, cosmetic, and pharmaceutical industries. They are not only responsible for capturing solar energy to carry out photosynthesis, but also play a role in photoprotective processes and display antioxidant activity, all of which contribute to effective biomass and oxygen production. Diatoms are organisms of a distinct pigment composition, substantially different from that present in plants. Apart from light-harvesting pigments such as chlorophyll a, chlorophyll c, and fucoxanthin, there is a group of photoprotective carotenoids which includes β-carotene and the xanthophylls, diatoxanthin, diadinoxanthin, violaxanthin, antheraxanthin, and zeaxanthin, which are engaged in the xanthophyll cycle. Additionally, some intermediate products of biosynthetic pathways have been identified in diatoms as well as unusual pigments, e.g., marennine. Marine algae have become widely recognized as a source of unique bioactive compounds for potential industrial, pharmaceutical, and medical applications. In this review, we summarize current knowledge on diatom photosynthetic pigments complemented by some new insights regarding their physico-chemical properties, biological role, and biosynthetic pathways, as well as the regulation of pigment level in the cell, methods of purification, and significance in industries. PMID:26389924

  8. The Photosynthetic Cycle

    DOE R&D Accomplishments Database

    Calvin, Melvin

    1955-03-21

    A cyclic sequence of transformations, including the carboxylation of RuDP (ribulose diphosphate) and its re-formation, has been deduced as the route for the creation of reduced carbon compounds in photosynthetic organisms. With the demonstration of RuDP as substrate for the carboxylation in a cell-free system, each of the reactions has now been carried out independently in vitro. Further purification of this last enzyme system has confirmed the deduction that the carboxylation of RuDP leads directly to the two molecules of PGA (phosphoglyceric acid) involving an internal dismutation and suggesting the name "carboxydismutase" for the enzyme. As a consequence of this knowledge of each of the steps in the photosynthetic CO{sub 2} reduction cycle, it is possible to define the reagent requirements to maintain it. The net requirement for the reduction of one molecule of CO{sub 2} is four equivalents of [H]and three molecules of ATP (adenine triphosphate). These must ultimately be supplied by the photochemical reaction. Some possible ways in which this may be accomplished are discussed.

  9. Photosynthetic Pigments in Diatoms

    PubMed Central

    Kuczynska, Paulina; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

    2015-01-01

    Photosynthetic pigments are bioactive compounds of great importance for the food, cosmetic, and pharmaceutical industries. They are not only responsible for capturing solar energy to carry out photosynthesis, but also play a role in photoprotective processes and display antioxidant activity, all of which contribute to effective biomass and oxygen production. Diatoms are organisms of a distinct pigment composition, substantially different from that present in plants. Apart from light-harvesting pigments such as chlorophyll a, chlorophyll c, and fucoxanthin, there is a group of photoprotective carotenoids which includes β-carotene and the xanthophylls, diatoxanthin, diadinoxanthin, violaxanthin, antheraxanthin, and zeaxanthin, which are engaged in the xanthophyll cycle. Additionally, some intermediate products of biosynthetic pathways have been identified in diatoms as well as unusual pigments, e.g., marennine. Marine algae have become widely recognized as a source of unique bioactive compounds for potential industrial, pharmaceutical, and medical applications. In this review, we summarize current knowledge on diatom photosynthetic pigments complemented by some new insights regarding their physico-chemical properties, biological role, and biosynthetic pathways, as well as the regulation of pigment level in the cell, methods of purification, and significance in industries. PMID:26389924

  10. A Novel R2R3-MYB Transcription Factor BpMYB106 of Birch (Betula platyphylla) Confers Increased Photosynthesis and Growth Rate through Up-regulating Photosynthetic Gene Expression

    PubMed Central

    Zhou, Chenguang; Li, Chenghao

    2016-01-01

    We isolated a R2R3-MYB transcription factor BpMYB106, which regulates photosynthesis in birch (Betula platyphylla Suk.). BpMYB106 mainly expresses in the leaf and shoot tip of birch, and its protein is localized in the nucleus. We further fused isolated a 1588 bp promoter of BpMYB106 and analyzed it by PLACE, which showed some cis-acting elements related to photosynthesis. BpMYB106 promoter β-glucuronidase (GUS) reporter fusion studies gene, the result, showed the GUS reporter gene in transgenic birch with BpMYB106 promoter showed strong activities in shoot tip, cotyledon margins, and mature leaf trichomes. The overexpression of BpMYB106 in birch resulted in significantly increased trichome density, net photosynthetic rate, and growth rate as compared with the wild-type birch. RNA-Seq profiling revealed the upregulation of several photosynthesis-related genes in the photosynthesis and oxidative phosphorylation pathways in the leaves of transgenic plants. Yeast one-hybrid analysis, coupled with transient assay in tobacco, revealed that BpMYB106 binds a MYB binding site MYB2 in differentially expressed gene promoters. Thus, BpMYB106 may directly activate the expression of a range of photosynthesis related genes through interacting with the MYB2 element in their promoters. Our study demonstrating the overexpression of BpMYB106—a R2R3-MYB transcription factor—upregulates the genes of the photosynthesis and oxidative phosphorylation pathways to improve photosynthesis. PMID:27047502

  11. Evolution of heliobacteria: implications for photosynthetic reaction center complexes

    NASA Technical Reports Server (NTRS)

    Vermaas, W. F.; Blankenship, R. E. (Principal Investigator)

    1994-01-01

    The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein show rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to the core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.

  12. Molecular cloning and expression of Neisseria meningitidis class 1 outer membrane protein in Escherichia coli K-12.

    PubMed Central

    Barlow, A K; Heckels, J E; Clarke, I N

    1987-01-01

    A genomic library of meningococcal DNA from a clinical isolate of Neisseria meningitidis was constructed in the expression vector lambda gt11. Outer membrane complex was prepared from the same strain and used to immunize rabbits to raise polyclonal anti-outer membrane complex serum. The amplified library was probed with this polyclonal serum, and seven expressing recombinants were isolated; further investigations indicated these to be identical. The expressed meningococcal gene in these recombinants was fused to vector beta-galactosidase and shown to encode epitopes present on the 42-kilodalton class 1 outer membrane protein. Estimation of the size of the recombinant fusion protein suggests that up to 40 kilodaltons of protein-coding sequence is present. The lambda gt11 recombinant contains a 3.4-kilobase DNA insert, which has been recloned into a plasmid and characterized by restriction endonuclease analysis. A restriction fragment from the insert, representing the protein-coding region hybridizes to a single 2.2-kilobase XbaI fragment from the homologous strain and to similar-sized XbaI fragments in other strains of meningococci, expressing antigenically distinct class 1 proteins. Images PMID:3117690

  13. Abnormal gene expression of proinflammatory cytokines and their membrane-bound receptors in the lymphocytes of depressed patients.

    PubMed

    Rizavi, Hooriyah S; Ren, Xinguo; Zhang, Hui; Bhaumik, Runa; Pandey, Ghanshyam N

    2016-06-30

    Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in plasma of depressed patients. In this study, we examined the role of cytokines and their membrane-bound receptors in major depressive disorder (MDD). We determined the protein and mRNA expression of proinflammatory cytokines, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and mRNA expression of their membrane-bound receptors in the lymphocytes from 31 hospitalized MDD patients and 30 non-hospitalized normal control (NC) subjects. The subjects were diagnosed according to DSM-IV criteria. Protein levels of cytokines were determined by ELISA, and mRNA levels in lymphocytes were determined by the qPCR method. We found that the mean mRNA levels of the proinflammatory cytokines IL-1β, IL-6, TNF-α, their receptors, TNFR1, TNFR2, IL-1R1 and the antagonist IL-1RA were significantly increased in the lymphocytes of MDD patients compared with NC. No significant differences in the lymphocyte mRNA levels of IL-1R2, IL-6R, and Gp130 were observed between MDD patients and NC. These studies suggest abnormal gene expression of these cytokines and their membrane-bound receptors in the lymphocytes of MDD patients, and that their mRNA expression levels in the lymphocytes could be a useful biomarker for depression. PMID:27138824

  14. Immunochemical Analysis of the Temporal and Tissue-Specific Expression of an Avena sativa Plasma Membrane Determinant 1

    PubMed Central

    Lynes, Michael A.

    1992-01-01

    An immunoglobulin Mk monoclonal (F8IVE9) antibody raised against oat (Avena sativa cv Garry) root homogenate has been produced and characterized. The predominant target bound by this antibody is a 62-kilodalton protein (p62) that is expressed in both oat root and oat shoot cells. Treatment of the oat antigen with periodate, or with recombinant N-glycanase, affects the F8IVE9 binding to the antigen, suggesting that the specific epitope for this monoclonal antibody involves a carbohydrate determinant. Levels of p62 present in cells of the oat root increase approximately twofold as the root tissue matures during the first 11 days postgermination. In contrast, levels of expression in shoot tissue remain relatively constant during the same period. The p62 antigen has been shown to be expressed at the plasma membrane by immunohistochemical means, by immunofluorescent labeling of protoplasts, and by enzyme-linked immunosorbent assay analysis of purified plasma membrane. The F8IVE9 antigenic target appears to be uniformly distributed through root tissue but is differentially expressed in specific sections of the shoot. F8IVE9 antibody also binds to antigens expressed in a number of other species, including clover, corn, pea, broccoli, mustard, and bean, and has been shown to bind to Samanea protoplast plasma membranes. This monoclonal antibody may prove to be useful for a variety of investigations, including an analysis of the specific patterns of cellular differentiation that occur during early morphogenesis, and the characterization of plasma membrane-associated elements in plants. ImagesFigure 3Figure 5Figure 7 PMID:16668621

  15. Molecular identification and differential expression of sensory neuron membrane proteins in the antennae of the black cutworm moth Agrotis ipsilon.

    PubMed

    Gu, Shao-Hua; Yang, Ruo-Nan; Guo, Meng-Bo; Wang, Gui-Rong; Wu, Kong-Ming; Guo, Yu-Yuan; Zhou, Jing-Jiang; Zhang, Yong-Jun

    2013-04-01

    The insect sensory neuron membrane proteins (SNMPs) SNMP1 and SNMP2 are transmembrane domain-containing proteins and are homologs of the vertebrate CD36 transmembrane proteins. It has been suggested that SNMPs play a significant role in insect chemoreception. Previous studies have demonstrated that SNMP1 is expressed in the pheromone-sensitive olfactory receptor neurons (ORNs), whereas SNMP2 is expressed in the supporting cells. In this study, we identified two full-length SNMP transcripts, AipsSNMP1 and AipsSNMP2, in the black cutworm moth Agrotis ipsilon (Hufnagel). The qRT-PCR results indicated that the AipsSNMP1 and AipsSNMP2 transcripts were expressed significantly higher in the antennae than in other tissues of both sexes. The expression of AipsSNMP1 and AipsSNMP2 in the antennae from different development stages of both sexes was investigated and was shown to begin to express in the pupae stage from 3days before emergence and then increased dramatically at the day of the emergence, and the high expression levels were maintained during the following 4days after the emergence in both sexes. The mating status had no effect on the expression levels of the AipsSNMP1 and AipsSNMP2 transcripts. Consistent with previous in situ hybridization studies in other Lepidoptera insects, our immunolocalization results at protein level demonstrated that both AipsSNMP1 and AipsSNMP2 were expressed in pheromone-sensitive sensilla trichodea but with a completely different expression profile. AipsSNMP1 is more uniformed and highly expressed along the membrane of the ORN dendrites, whereas AipsSNMP2 is widely distributed at the bottom of the sensilla trichodea and highly localized in the sensillum lymph. Our studies provide further detailed evidence for the involvement and general functional role of insect SNMPs in the detection of sex pheromones and general odorant molecules. PMID:23454276

  16. Membrane-attached Cytokines Expressed by mRNA Electroporation Act as Potent T-Cell Adjuvants.

    PubMed

    Weinstein-Marom, Hadas; Pato, Aviad; Levin, Noam; Susid, Keren; Itzhaki, Orit; Besser, Michal J; Peretz, Tamar; Margalit, Alon; Lotem, Michal; Gross, Gideon

    2016-01-01

    Proinflammatory cytokines are widely explored in different adoptive cell therapy protocols for enhancing survival and function of the transferred T cells, but their systemic administration is often associated with severe toxicity which limits their clinical use. To confine cytokine availability to the therapeutic T cells, we expressed 3 key cytokines, IL-2, IL-12, and IL-15, as integral T-cell membrane proteins. To prevent permanent activation of growth signaling pathways, we delivered these genes to T cells through mRNA electroporation. The engineered cytokines could be detected on the surface of mRNA-transfected cells and binding to their cell-surface receptors mainly occurred in cis. The 3 human cytokines supported the ex vivo growth of activated human CD8 and CD4 T cells for at least 6 days posttransfection, comparably to high-dose soluble IL-2. Similarly, membrane IL-2, membrane IL-12, and, to a lesser extent, membrane IL-15, were comparable with their soluble counterparts in supporting proliferation of splenic mouse CD8 T cells. Following electroporation of human CD8 T cells and antimelanoma tumor-infiltrating lymphocytes, membrane cytokines synergized with constitutively active toll-like receptor 4 in inducing interferon-γ secretion. Efficient cooperation with TLR4 was also evident in the upregulation of the activation molecules CD25, CD69, CD137 (4-1BB), and CD134 (OX40). Taken together, membrane cytokines expressed through mRNA transfection emerge as effective tools for enhancing T-cell proliferation and function and may have potential use in adoptive T-cell therapy. PMID:26849075

  17. Overcoming the toxicity of membrane peptide expression in bacteria by upstream insertion of Asp-Pro sequence.

    PubMed

    Montigny, Cédric; Penin, François; Lethias, Claire; Falson, Pierre

    2004-01-28

    Transmembrane (TM) peptides often induce toxic effects when expressed in bacteria, probably due to membrane destabilization. We report here that in the case of the TM domains of hepatitis C virus (HCV) E1 and E2 envelope proteins, which are both particularly toxic for the bacteria, the insertion of the Asp-Pro (DP) sequence dramatically reduced their toxicities and promoted their expressions when produced as glutathione S-transferase (GST) GST-DP-TM chimeras. Subcellular fractionation showed that these chimeras co-sediment with the membrane fraction and contain active GST that could be solubilized with a mild detergent. Surprisingly, immuno-gold electron microscopy clearly showed that such chimeras are not localized in the membrane but in the cytosol. We thus postulate that they likely form proteo-lipidic aggregates, which prevent the bacteria from toxicity by sequestering the TM part of the chimeras. The reduction of toxicity in the presence of the Asp-Pro sequence is possibly due to Asp's negative charge that probably disadvantages the binding of the TM peptides to the membrane. In addition, the structural features of Pro residue could promote the formation of chimera aggregates. PMID:14757220

  18. Isotopically Labeled Expression in E. coli, Purification, and Refolding of the Full Ectodomain of the Influenza Virus Membrane Fusion Protein

    PubMed Central

    Curtis-Fisk, Jaime; Spencer, Ryan M.; Weliky, David P.

    2008-01-01

    This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of E. coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A600 ~ 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the 13CO chemical shift measured using solid-state nuclear magnetic resonance spectroscopy. PMID:18640277

  19. A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots.

    PubMed Central

    Santoni, Véronique; Vinh, Joëlle; Pflieger, Delphine; Sommerer, Nicolas; Maurel, Christophe

    2003-01-01

    Aquaporins are channel proteins that facilitate the diffusion of water across cell membranes. The genome of Arabidopsis thaliana encodes 35 full-length aquaporin homologues. Thirteen of them belong to the plasma membrane intrinsic protein (PIP) subfamily and predominantly sit at the plasma membrane (PM). In the present work we combine separations of membrane proteins (by one- and two-dimensional gel electrophoresis) with identification by MS (matrix-assisted laser-desorption ionization-time-of-flight and electrospray-ionization tandem MS) to take an inventory of aquaporin isoforms expressed in the PM of Arabidopsis thaliana roots. Our analysis provides direct evidence for the expression of five PIPs (PIP1;1, PIP1;5, PIP2;1, PIP2;2 and PIP2;7) in the root PM and suggests the presence of at least three other PIP isoforms. In addition, we show that the same PIP isoform can be present under several forms with distinct isoelectric points. More specifically, we identify phosphorylated aquaporins in the PIP1 and PIP2 subgroups and suggest the existence of other post-translational modifications. Their identification should provide clues to reveal novel molecular mechanisms for aquaporin regulation. PMID:12678916

  20. A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins.

    PubMed

    Kleinlogel, Sonja; Terpitz, Ulrich; Legrum, Barbara; Gökbuget, Deniz; Boyden, Edward S; Bamann, Christian; Wood, Phillip G; Bamberg, Ernst

    2011-12-01

    The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin. PMID:22056675

  1. Prebiotic photosynthetic reactions.

    PubMed

    Chittenden, G J; Schwartz, A W

    1981-01-01

    Historically, numerous attempts have been made to mimic - by means of inorganic model reactions - the photosynthetic fixation of CO2 by green plants. The literature in this field is strewn with claims and counter-claims. Two factors have led us to reexamine this subject: firstly; doubts concerning the highly reducing model for the atmosphere of the primitive Earth and secondly; recent results which demonstrate that photoreductive fixation is feasable on a suitable catalytic surface, for both CO2 and N2. The latter observation is of particular interest due to the well-known susceptibility of NH3 to photolytic destruction. Our review of the literature leads us to suggest that similar processes would have been plausible for the primitive Earth and could have been prebiotic precursors to an early development of CO2-fixing autotrophs. PMID:6791723

  2. Photosynthetic light reactions: integral to chloroplast retrograde signalling.

    PubMed

    Gollan, Peter J; Tikkanen, Mikko; Aro, Eva-Mari

    2015-10-01

    Chloroplast retrograde signalling is ultimately dependent on the function of the photosynthetic light reactions and not only guides the acclimation of the photosynthetic apparatus to changing environmental and metabolic cues, but has a much wider influence on the growth and development of plants. New information generated during the past few years about regulation of photosynthetic light reactions and identification of the underlying regulatory proteins has paved the way towards better understanding of the signalling molecules produced in chloroplasts upon changes in the environment. Likewise, the availability of various mutants lacking regulatory functions has made it possible to address the role of excitation energy distribution and electron flow in the thylakoid membrane in inducing the retrograde signals from chloroplasts to the nucleus. Such signalling molecules also induce and interact with hormonal signalling cascades to provide comprehensive information from chloroplasts to the nucleus. PMID:26318477

  3. 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

    Thomas, Peter; Pang, Yefei; Dong, Jing

    2014-03-01

    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 [(3)H]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

  4. Expression of the moss PpLEA4-20 gene in rice enhances membrane protection and client proteins stability.

    PubMed

    Li, Li; Deng, Dandan; Chen, Xi; Wu, Baomei; Hu, Ke; Qiu, Tianhang; Cui, Suxia; Huang, Fang

    2015-05-01

    Green vegetative tissues of the moss Physcomitrella patens possess a powerful ability to tolerate severe drought stress. Proteomics analysis have revealed that a large number of late embryogenesis abundant (LEA) proteins were key players in the drought tolerance of the photosynthetic tissues. PpLEA4-20, a member of the moss LEA protein family, was selected for further function study using an ectopic expression method in rice. Through molecular identification via PCR, southern blotting and TAIL-PCR, we demonstrated that the PpLEA4-20 gene was transformed and inserted into a non-encoded region in chromosome 4 of rice and expressed stably in transgenic rice. Unexpectedly, PpLEA4-20 protein emerged as two high-expressed spots on 2-D gels generated from transgenic rice, suggesting that PpLEA4-20 proteins are complete compatible and might be modified in rice. Both growth and physiological analysis showed that seedlings of transgenic PpLEA4-20 rice displayed altered phenotypes and tolerance to salt. In addition, electrolyte leakage was reduced in transgenic PpLEA4-20 compared to wild type under stress conditions. Anti-aggregation analysis found that the PpLEA4-20 protein expressed in rice remained soluble at high temperature and in addition to some native proteins from transgenic PpLEA4-20 rice. Based on Nano LC MS/MS analysis, we identified several proteins from transgenic PpLEA4-20 rice of increased heat-stability. Our results provide evidence for a role of PpLEA4-20 in salt tolerance and stabilization of client proteins. PMID:25791479

  5. Characterization of a multiple endogenously expressed Adenosine triphosphate-Binding Cassette transporters using nuclear and cellular membrane affinity chromatography columns

    PubMed Central

    Khadeer, M.A.; Shimmo, R.; Wainer, I.W.; Moaddel, R.

    2014-01-01

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of 8 compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN229) column and decreased it (−5%) on the NMAC(LN229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

  6. Cytokinin membrane receptors participate in regulation of plastid genome expression in the skotomorphogenesis.

    PubMed

    Doroshenko, A S; Danilova, M N; Kudryakova, N V; Soloviev, A A; Kusnetsov, V V

    2016-07-01

    Analysis by real-time PCR of single and double insertion mutants of A. thaliana with inactivated cytokinin receptor genes showed that the level of transcripts of some of plastid genes during skotomorphogenesis depended on the state of functionally active receptor AHK3. The cytokinin-regulated plastid encoded genes involved the housekeeping genes (rpoB and accD) and the genes for photosynthetic proteins (ndhA, psbA, atpB, and psaA). However, the absence of hormone activation of plastid encoded genes engaged in the translation of plastid proteins, rRNA (rrn16), and tRNA (trnE), indirectly indicate the disruption of the synthesis of chloroplast proteins in the dark. PMID:27599515

  7. Maximum photosynthetic efficiency of biomass growth: a criticism of some measurements

    SciTech Connect

    Lee, Y.K.; Pirt, S.J.

    1982-02-01

    The yield of biomass produced in a photosynthetic culture is an expression of the photosynthetic efficiency. Microbial cells consume energy for both growth and for maintenance. The bioenergetics of Chlorella cultures and the maximum growth yields obtained by various researchers are examined in this paper.

  8. The effects of down-regulating expression of Arabidopsis thaliana membrane-associated acyl-CoA binding protein 2 on acyl-lipid composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple classes of acyl-CoA binding proteins are encoded by plant genomes, including a plant-unique class of predicted integral membrane-proteins. Transcript analysis revealed that both of the integral membrane-acyl-CoA binding proteins of Arabidopsis thaliana, ACBP1 and ACBP2, are expressed in al...

  9. Channel catfish (Ictalurus punctatus Rafinesque, 1818) tetraspanin membrane protein family: Identification, characterization, and expression analysis of CD63 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CD63, known as lysosome associated membrane protein 3 (LAMP-3), is a member of the tetraspanin integral membrane protein family. This protein plays many important roles in immuno-physiological functions. In this communication, we report the identification, characterization, and expression analysis...

  10. Protein Disulfide Isomerase Chaperone ERP-57 Decreases Plasma Membrane Expression of the Human GnRH Receptor

    PubMed Central

    Yánez, Rodrigo Ayala; Conn, P. Michael

    2012-01-01

    Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX) which interact and restrict plasma membrane expression of gonadotropin releasing hormone receptor (GnRHR), a G protein coupled receptor. CANX also interacts with ERP-57, a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57, promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (Cys14-Cys200 and Cys114-Cys196), that, when broken, its expression at plasma membrane decreases. To determine if the presence of chaperones CANX and ERP-57 exert an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken bridges (Cys→Ala) along with the wild type hGnRHR. The effect of chaperones on mutants was insignificant, whereas the overexpression of ERP-57 led to a wild type hGnRHR retention which was further enhanced by cotransfection with CANX cDNA disclosing receptor retention by ERP-57 augmented by CANX, suggesting a quality control mechanism. PMID:20029959

  11. Pseudomonas aeruginosa Cell Membrane Protein Expression from Phenotypically Diverse Cystic Fibrosis Isolates Demonstrates Host-Specific Adaptations.

    PubMed

    Kamath, Karthik Shantharam; Pascovici, Dana; Penesyan, Anahit; Goel, Apurv; Venkatakrishnan, Vignesh; Paulsen, Ian T; Packer, Nicolle H; Molloy, Mark P

    2016-07-01

    Pseudomonas aeruginosa is a Gram-negative, nosocomial, highly adaptable opportunistic pathogen especially prevalent in immuno-compromised cystic fibrosis (CF) patients. The bacterial cell surface proteins are important contributors to virulence, yet the membrane subproteomes of phenotypically diverse P. aeruginosa strains are poorly characterized. We carried out mass spectrometry (MS)-based proteome analysis of the membrane proteins of three novel P. aeruginosa strains isolated from the sputum of CF patients and compared protein expression to the widely used laboratory strain, PAO1. Microbes were grown in planktonic growth condition using minimal M9 media, and a defined synthetic lung nutrient mimicking medium (SCFM) limited passaging. Two-dimensional LC-MS/MS using iTRAQ labeling enabled quantitative comparisons among 3171 and 2442 proteins from the minimal M9 medium and in the SCFM, respectively. The CF isolates showed marked differences in membrane protein expression in comparison with PAO1 including up-regulation of drug resistance proteins (MexY, MexB, MexC) and down-regulation of chemotaxis and aerotaxis proteins (PA1561, PctA, PctB) and motility and adhesion proteins (FliK, FlgE, FliD, PilJ). Phenotypic analysis using adhesion, motility, and drug susceptibility assays confirmed the proteomics findings. These results provide evidence of host-specific microevolution of P. aeruginosa in the CF lung and shed light on the adaptation strategies used by CF pathogens. PMID:27246823

  12. GlnD is essential for NifA activation, NtrB/NtrC-regulated gene expression, and posttranslational regulation of nitrogenase activity in the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum.

    PubMed

    Zhang, Yaoping; Pohlmann, Edward L; Roberts, Gary P

    2005-02-01

    GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme and is thought to be the primary sensor of nitrogen status in the cell. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) proteins, which in turn regulate a variety of other proteins. We report here the characterization of glnD mutants from the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum and the analysis of the roles of GlnD in the regulation of nitrogen fixation. Unlike glnD mutations in Azotobacter vinelandii and some other bacteria, glnD deletion mutations are not lethal in R. rubrum. Such mutants grew well in minimal medium with glutamate as the sole nitrogen source, although they grew slowly with ammonium as the sole nitrogen source (MN medium) and were unable to fix N(2). The slow growth in MN medium is apparently due to low glutamine synthetase activity, because a DeltaglnD strain with an altered glutamine synthetase that cannot be adenylylated can grow well in MN medium. Various mutation and complementation studies were used to show that the critical uridylyltransferase activity of GlnD is localized to the N-terminal region. Mutants with intermediate levels of uridylyltransferase activity are differentially defective in nif gene expression, the posttranslational regulation of nitrogenase, and NtrB/NtrC function, indicating the complexity of the physiological role of GlnD. These results have implications for the interpretation of results obtained with GlnD in many other organisms. PMID:15687189

  13. Nanoscale Optoelectronic Photosynthetic Devices

    NASA Astrophysics Data System (ADS)

    Greenbaum, Elias; Lee, Ida; Guillorn, Michael; Lee, James W.; Simpson, Michael L.

    2001-03-01

    This presentation provides an overview and recent progress in the Oak Ridge National Laboratory research program in molecular electronics and green plant photosynthesis. The photosynthetic reaction center is a nanoscale molecular diode and photovoltaic device. The key thrust of our research program is the construction of molecular electronic devices from these nanoscale structures. Progress in this multidisciplinary research program has been demonstrated by direct electrical contact of emergent electrons with the Photosystem I (PS I) reaction center by nanoparticle precipitation. Demonstration of stable diode properties of isolated reaction centers combined with the ability to orient PS I by self-assembly on a planar surface, makes this structure a good building block for 2-D and potentially 3-D devices. Metallization of isolated PS I does not alter their fundamental photophysical properties and they can be bonded to metal surfaces. We report here the first measurement of photovoltage from single PS I reaction centers. Working at the Cornell University National Nanofabrication Facility, we have constructed sets of dissimilar metal electrodes separated by distances as small as 6 nm. We plan to use these structures to make electrical contact to both ends of oriented PSI reaction centers and thereby realize biomolecular logic circuits. Potential applications of PSI reaction centers for optoelectronic applications as well as molecular logic device construction will be discussed.

  14. Loss of membranous VEGFR1 expression is associated with an adverse phenotype and shortened survival in breast cancer.

    PubMed

    Lebok, Patrick; Huber, Julia; Burandt, Eike-Christian; Lebeau, Annette; Marx, Andreas Holger; Terracciano, Luigi; Heilenkötter, Uwe; Jänicke, Fritz; Müller, Volkmar; Paluchowski, Peter; Geist, Stefan; Wilke, Christian; Simon, Ronald; Sauter, Guido; Quaas, Alexander

    2016-08-01

    Angiogenesis is a key process in tumor growth and progression, which is controlled by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). In order to better understand the prevalence and prognostic value of VEGFR1 expression in breast cancer, a tissue microarray containing >2,100 breast cancer specimens, with clinical follow‑up data, was analyzed by immunohistochemistry using an antibody directed against the membrane‑bound full‑length receptor protein. The results demonstrated that membranous VEGFR1 staining was detected in all (5 of 5) normal breast specimens. In carcinoma specimens, membranous staining was negative in 3.1%, weak in 6.3%, moderate in 10.9%, and strong in 79.7% of the 1,630 interpretable tissues. Strong staining was significantly associated with estrogen receptor and progesterone receptor expression, but was inversely associated with advanced tumor stage (P=0.0431), high Bloom-Richardson-Ellis Score for Breast Cancer grade and low Ki67 labeling index (both P<0.0001). Cancers with moderate to strong (high) VEGFR1 expression were associated with significantly improved overall survival, as compared with tumors exhibiting negative or weak (low) expression (P=0.0015). This association was also detected in the subset of nodal‑positive cancers (P=0.0018), and in the subset of 185 patients who had received tamoxifen as the sole therapy (P=0.001). In conclusion, these data indicated that membrane‑bound VEGFR1 is frequently expressed in normal and cancerous breast epithelium. In addition, reduced or lost VEGFR1 expression may serve as a marker for poor prognosis in patients with breast cancer, who might not optimally benefit from endocrine therapy. PMID:27357606

  15. Micromachined microbial and photosynthetic fuel cells

    NASA Astrophysics Data System (ADS)

    Chiao, Mu; Lam, Kien B.; Lin, Liwei

    2006-12-01

    This paper presents two types of fuel cells: a miniature microbial fuel cell (µMFC) and a miniature photosynthetic electrochemical cell (µPEC). A bulk micromachining process is used to fabricate the fuel cells, and the prototype has an active proton exchange membrane area of 1 cm2. Two different micro-organisms are used as biocatalysts in the anode: (1) Saccharomyces cerevisiae (baker's yeast) is used to catalyze glucose and (2) Phylum Cyanophyta (blue-green algae) is used to produce electrons by a photosynthetic reaction under light. In the dark, the µPEC continues to generate power using the glucose produced under light. In the cathode, potassium ferricyanide is used to accept electrons and electric power is produced by the overall redox reactions. The bio-electrical responses of µMFCs and µPECs are characterized with the open-circuit potential measured at an average value of 300-500 mV. Under a 10 ohm load, the power density is measured as 2.3 nW cm-2 and 0.04 nW cm-2 for µMFCs and µPECs, respectively.

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

    PubMed

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

    1986-09-01

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

  17. Inflammatory Signalling in Fetal Membranes: Increased Expression Levels of TLR 1 in the Presence of Preterm Histological Chorioamnionitis

    PubMed Central

    Waring, Gareth J.; Robson, Stephen C.; Bulmer, Judith N.; Tyson-Capper, Alison J.

    2015-01-01

    Histological chorioamnionitis (HCA) is an established marker of ascending infection, a major cause of preterm birth. No studies have characterised the global change in expression of genes involved in the toll-like receptor (TLR) signalling pathways in the presence of HCA in the setting of preterm birth (pHCA). Fetal membranes were collected immediately after delivery and underwent histological staging for inflammation to derive 3 groups; term spontaneous labour without HCA (n = 9), preterm birth <34 weeks gestation without HCA (n = 8) and pHCA <34 weeks (n = 12). Profiling arrays ran in triplicate for each group were used to determine the expression of 84 genes associated with TLR signalling and screen for genes of interest (fold change >2; p<0.1). Expression of identified genes was validated individually for all samples, relative to GAPDH, using RT-PCR. Expression of TLR 1, TLR 2, lymphocyte antigen 96, interleukin 8 and Interleukin-1 receptor-associated kinase-like 2 was increased in pHCA (p<0.05). Degree of expression was positively associated with histological staging of both maternal and fetal inflammation (p<0.05). The inflammatory expression profile at the maternal/fetal interface associated with pHCA, a reflection of ascending infection, is extremely heterogeneous suggesting polymicrobial involvement with activation of a common pathway. Antagonism of TLR 1 and TLR 2 signalling in this setting warrants further assessment. PMID:25965269

  18. Effect of clinostating on photosynthetic apparatus of pea plants

    NASA Astrophysics Data System (ADS)

    Kochubey, S. M.; Volovik, O. I.; Porubleva, L. V.; Shevchenko, V. V.

    The photosynthetic membrane composition and low temperature fluorescence spectra were analyzed for pea chloroplasts from control and clinostated plants. Clinorotation induces a decrease in the amount of the oligomeric form of the light-harvesting chlorophyll a/b complex (LHCII) and an increase of its monomeric form. Some changes in organization of photosystem 1 (PS1) complex were revealed as well. These changes are in accordance with the variations of fluorescence characteristics and photochemical activity.

  19. Specific Glycosylation of Membrane Proteins in Epithelial Ovarian Cancer Cell Lines: Glycan Structures Reflect Gene Expression and DNA Methylation Status *

    PubMed Central

    Anugraham, Merrina; Jacob, Francis; Nixdorf, Sheri; Everest-Dass, Arun Vijay; Heinzelmann-Schwarz, Viola; Packer, Nicolle H.

    2014-01-01

    Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. N-glycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the “bisecting N-acetyl-glucosamine” type N-glycans, increased levels of α 2–6 sialylated N-glycans and “N,N′-diacetyl-lactosamine” type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique “bisecting GlcNAc” type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association

  20. Specific glycosylation of membrane proteins in epithelial ovarian cancer cell lines: glycan structures reflect gene expression and DNA methylation status.

    PubMed

    Anugraham, Merrina; Jacob, Francis; Nixdorf, Sheri; Everest-Dass, Arun Vijay; Heinzelmann-Schwarz, Viola; Packer, Nicolle H

    2014-09-01

    Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. N-glycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the "bisecting N-acetyl-glucosamine" type N-glycans, increased levels of α 2-6 sialylated N-glycans and "N,N'-diacetyl-lactosamine" type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique "bisecting GlcNAc" type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association with epigenetic

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

    PubMed

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

    2015-02-01

    To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na⁺-K⁺ ATPase. Assuming that Na⁺-K⁺ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r² > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs. PMID:25488931

  2. Peroxiredoxin-2 expression is increased in β-thalassemic mouse red cells but is displaced from the membrane as a marker of oxidative stress

    PubMed Central

    Matte, Alessandro; Low, Philip S.; Turrini, Franco; Bertoldi, Mariarita; Campanella, Maria Estela; Spano, Daniela; Pantaleo, Antonella; Siciliano, Angela; De Franceschi, Lucia

    2012-01-01

    Peroxiredoxin 2 (Prx2), the third most abundant cytoplasmic protein in red blood cells (RBCs), is involved in the defense against oxidative stress. Although much is known about Prx2 in healthy RBCs, its role in pathological RBCs remains largely unexplored. Here, we show that the expression and net content of Prx2 are markedly increased in RBCs from two mouse models of β-thalassemia (β-thal; Hbbth/th and Hbbth3/+ strains). We also demonstrate that the increased expression of Prx2 correlates with the severity of the disease and that the amount of Prx2 bound to the membrane is markedly reduced in β-thal mouse RBCs. To explore the impact of oxidative stress on Prx2 membrane association, we examined Prx2 dimerization and membrane translocation in murine RBCs exposed to various oxidants (phenylhydrazine, PHZ; diamide; H2O2). PHZ-treated RBCs, which mimic the membrane damage in β-thal RBCs, exhibited a kinetic correlation among Prx2 membrane displacement, intracellular methemoglobin levels, and hemichrome membrane association, suggesting the possible masking of Prx2 docking sites by membrane-bound hemichromes, providing a possible mechanism for the accumulation of oxidized/dimerized Prx2 in the cytoplasm and the increased membrane damage in β-thal RBCs. Thus, reduced access of Prx2 to the membrane in β-thal RBCs represents a new factor that could contribute to the oxidative damage characterizing the pathology. PMID:20488244

  3. Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

    PubMed

    Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

    2009-10-01

    The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions. PMID:19602722

  4. Photosynthetic Machineries in Nano-Systems

    PubMed Central

    Nagy, László; Magyar, Melinda; Szabó, Tibor; Hajdu, Kata; Giotta, Livia; Dorogi, Márta; Milano, Francesco

    2014-01-01

    Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bionanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e.g., photovoltaics) or biosensing (e.g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field. PMID:24678673

  5. Structural basis of photosynthetic water-splitting

    SciTech Connect

    Shen, Jian-Ren; Kawakami, Keisuke; Kamiya, Nobuo

    2013-12-10

    Photosynthetic water-splitting takes place in photosystem II (PSII), a membrane protein complex consisting of 20 subunits with an overall molecular mass of 350 kDa. The light-induced water-splitting reaction catalyzed by PSII not only converts light energy into biologically useful chemical energy, but also provides us with oxygen indispensible for sustaining oxygenic life on the earth. We have solved the structure of PSII at a 1.9 Å resolution, from which, the detailed structure of the Mn{sub 4}CaO{sub 5}-cluster, the catalytic center for water-splitting, became clear. Based on the structure of PSII at the atomic resolution, possible mechanism of light-induced water-splitting was discussed.

  6. Differential Allocation to Photosynthetic and Non-Photosynthetic Nitrogen Fractions among Native and Invasive Species

    PubMed Central

    Funk, Jennifer L.; Glenwinkel, Lori A.; Sack, Lawren

    2013-01-01

    Invasive species are expected to cluster on the “high-return” end of the leaf economic spectrum, displaying leaf traits consistent with higher carbon assimilation relative to native species. Intra-leaf nitrogen (N) allocation should support these physiological differences; however, N biochemistry has not been examined in more than a few invasive species. We measured 34 leaf traits including seven leaf N pools for five native and five invasive species from Hawaii under low irradiance to mimic the forest understory environment. We found several trait differences between native and invasive species. In particular, invasive species showed preferential N allocation to metabolism (amino acids) rather than photosynthetic light reactions (membrane-bound protein) by comparison with native species. The soluble protein concentration did not vary between groups. Under these low irradiance conditions, native species had higher light-saturated photosynthetic rates, possibly as a consequence of a greater investment in membrane-bound protein. Invasive species may succeed by employing a wide range of N allocation mechanisms, including higher amino acid production for fast growth under high irradiance or storage of N in leaves as soluble protein or amino acids. PMID:23700483

  7. Molecular cloning and expression of channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59 gene was cloned and analyzed. Total RNA from tissues was isolated and cDNA libraries were constructed by the rapid amplification cDNA end (RACE) method. The gene-specific primers in conjunction with the RAC...

  8. Developmental changes in milk fat globule membrane proteome expression during the transition from colostrum to milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shotgun Proteomics, using amine-reactive isobaric tags (iTRAQ) was used to quantify protein changes in milk fat globule membranes (MFGM) that were isolated from day 1 colostrum and compared to MFGM from day 7 milk. Eight Holstein cows were randomly assigned to 2 groups of 4 cow sample pools for a s...

  9. MOLECULAR CLONING AND EXPRESSION OF CHANNEL CATFISH, ICTALURUS PUNCTATUS, COMPLEMENT MEMBRANE ATTACK COMPLEX INHIBITOR CD59

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59 gene was cloned and analyzed. Total RNA from tissues was isolated and cDNA libraries were constructed by the rapid amplification cDNA end (RACE) method. The gene-specific primers in conjunction with the RAC...

  10. Differential Expression in Phanerochaete chrysosporium of Membrane-Associated Proteins Relevant to Lignin Degradation

    SciTech Connect

    Shary, Semarjit; Kapich, Alexander N.; Panisko, Ellen A.; Magnuson, Jon K.; Cullen, Dan; Hammel, Ken

    2008-10-02

    Fungal lignin-degrading systems must include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, secretion of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Despite their importance, little is known about the nature or regulation of these membrane-associated components. We grew the white rot basidiomycete Phanerochaete chrysosporium on cellulose or glucose as the carbon source and monitored the mineralization of a 14C-labeled synthetic lignin by these cultures to assess their ligninolytic competence. The results showed that the cellulose-grown cultures were ligninolytic, whereas the glucose-grown ones were not. We isolated microsomal membrane fractions from both types of culture and analyzed tryptic digests of them by shotgun liquid chromatography/tandem mass spectrometry. Comparison of the results against the predicted P. chrysosporium proteome showed that a catalase (Joint Genome Institute P. chrysosporium protein I.D. 124398), an alcohol oxidase (126879), two transporters (137220 and 132234), and two cytochrome P450s (5011 and 8912) were up-regulated under ligninolytic conditions. Real time reverse transcription polymerase chain reaction assays showed that RNA transcripts encoding all of these proteins were also up-regulated in ligninolytic cultures. Catalase 124398, alcohol oxidase 126879, and transporter 137220 were found in a proteomic analysis of partially purified plasma membranes from ligninolytic P. chrysosporium, and are therefore most likely associated with the outer envelope of the fungus.

  11. PLACENTAL EXPRESSION OF THE MEMBRANE FORM OF FOLATE BINDING PROTEIN DURING PREGNANCY IN SWINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous experiments indicated that secreted (s) and membrane (m) forms of folate binding protein (FBP) are present in the intrauterine environment of the pig. Our studies indicated that the two forms were produced sequentially; the secreted form was present in the intrauterine glands until Day 20 o...

  12. LLC-PK sub 1 cells express Na sup + -lactate cotransport in apical membranes after confluency

    SciTech Connect

    Poustis-Delpont, C.; Mengual, R.; Sudaka, P. )

    1988-12-01

    L-({sup 3}H)lactate uptake was characterized in LLC-PK{sub 1} cell apical membrane vesicles obtained by intensive culture on microcarrier beads. The apical membrane preparation technique involved MgCl{sub 2} precipitation. Na{sup +}-dependent L-({sup 3}H)lactate uptake was present only after confluency; its appearance paralleled the subcellular localization of aminopeptidase in apical membranes. L-({sup 3}H)lactate uptake was Na{sup +}-dependent and electrogenic. Only the Na{sup +}-dependent component of L({sup 3}H)lactate uptake was saturable with one family of independent carriers. The apparent affinity constant was 1.1 {plus minus} 0.25 mM and the apparent maximal velocity was 29 {plus minus} 3 nmol{center dot}mg{sup {minus}1}{center dot}min{sup {minus}1}. The Na{sup +}-lactate cotransport stoichiometry was 2 Na{sup +} for 1 lactate. The specificity of the L-lactate transport system was compatible with that of the monocarboxylic acid pathway described previously brush-border membranes of kidney cortex and discrete from the tricarboxylic acid carrier, the D-glucose transporter, and the general pathway for anions. The LLC-PK{sub 1} cell line appears to be a useful tool for study of the regulation of L-lactate uptake and biosynthesis of the renal monocarboxylic acid transporter.

  13. KCNJ10 (Kir4.1) is expressed in the basolateral membrane of the cortical thick ascending limb.

    PubMed

    Zhang, Chengbiao; Wang, Lijun; Su, Xiao-Tong; Lin, Dao-Hong; Wang, Wen-Hui

    2015-06-01

    The aim of the present study is to examine the role of Kcnj10 (Kir.4.1) in contributing to the basolateral K conductance in the cortical thick ascending limb (cTAL) using Kcnj10(+/+) wild-type (WT) and Kcnj10(-/-) knockout (KO) mice. The patch-clamp experiments detected a 40- and an 80-pS K channel in the basolateral membrane of the cTAL. Moreover, the probability of finding the 40-pS K was significantly higher in the late part of the cTAL close to the distal convoluted tubule than those in the initial part. Immunostaining showed that Kcnj10 staining was detected in the basolateral membrane of the cTAL but the expression was not uniformly distributed. The disruption of Kcnj10 completely eliminated the 40-pS K channel but not the 80-pS K channel, suggesting the role of Kcnj10 in forming the 40-pS K channel of the cTAL. Also, the disruption of Kcnj10 increased the probability of finding the 80-pS K channel in the cTAL, especially in the late part of the cTAL. Because the channel open probability of the 80-pS K channel in KO was similar to those of WT mice, the increase in the 80-pS K channel may be achieved by increasing K channel number. The whole cell recording further showed that K reversal potential measured with 5 mM K in the bath and 140 mM K in the pipette was the same in the WT and KO mice. Moreover, Western blot and immunostaining showed that the disruption of Kcnj10 did not affect the expression of Na-K-Cl cotransporter 2 (NKCC2). We conclude that Kir.4.1 is expressed in the basolateral membrane of cTAL and that the disruption of Kir.4.1 has no significant effect on the membrane potential of the cTAL and NKCC2 expression. PMID:25834074

  14. Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

    PubMed

    Bakari, Sana; Lembrouk, Mehdi; Sourd, Laura; Ousalem, Fares; André, François; Orlowski, Stéphane; Delaforge, Marcel; Frelet-Barrand, Annie

    2016-04-01

    Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies. PMID:26961909

  15. Photosynthetic production of itaconic acid in Synechocystis sp. PCC6803.

    PubMed

    Chin, Taejun; Sano, Mei; Takahashi, Tetsuya; Ohara, Hitomi; Aso, Yuji

    2015-02-10

    Here, we report the photosynthetic production of itaconic acid (IA), a promising building block, from carbon dioxide (CO₂) by Synechocystis sp. PCC6803. The engineered PCC6803 strain expressing cis-aconitate decarboxylase, the key enzyme in IA biosynthesis, produced 0.9 mg/L and 14.5 mg/L of IA at production rates of 42.8 μgL(-1)day(-1) and 919.0 μgL(-1)day(-1), under conditions of constant bubbling with air and 5% CO₂, respectively. This is the first report on the possibility of IA production from CO₂ via the photosynthetic process in cyanobacteria. PMID:25554635

  16. Role of membrane glycerolipids in photosynthesis, thylakoid biogenesis and chloroplast development.

    PubMed

    Kobayashi, Koichi

    2016-07-01

    The lipid bilayer of the thylakoid membrane in plant chloroplasts and cyanobacterial cells is predominantly composed of four unique lipid classes; monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). MGDG and DGDG are uncharged galactolipids that constitute the bulk of thylakoid membrane lipids and provide a lipid bilayer matrix for photosynthetic complexes as the main constituents. The glycolipid SQDG and phospholipid PG are anionic lipids with a negative charge on their head groups. SQDG and PG substitute for each other to maintain the amount of total anionic lipids in the thylakoid membrane, with PG having indispensable functions in photosynthesis. In addition to biochemical studies, extensive analyses of mutants deficient in thylakoid lipids have revealed important roles of these lipids in photosynthesis and thylakoid membrane biogenesis. Moreover, recent studies of Arabidopsis thaliana suggest that thylakoid lipid biosynthesis triggers the expression of photosynthesis-associated genes in both the nucleus and plastids and activates the formation of photosynthetic machineries and chloroplast development. Meanwhile, galactolipid biosynthesis is regulated in response to chloroplast functionality and lipid metabolism at transcriptional and post-translational levels. This review summarizes the roles of thylakoid lipids with their biosynthetic pathways in plants and discusses the coordinated regulation of thylakoid lipid biosynthesis with the development of photosynthetic machinery during chloroplast biogenesis. PMID:27114097

  17. Reduced expression of plasma membrane calcium ATPase 2 and collapsin response mediator protein 1 promotes death of spinal cord neurons.

    PubMed

    Kurnellas, M P; Li, H; Jain, M R; Giraud, S N; Nicot, A B; Ratnayake, A; Heary, R F; Elkabes, S

    2010-09-01

    The mechanisms underlying neuronal pathology and death in the spinal cord (SC) during inflammation remain elusive. We previously showed the important role of plasma membrane calcium ATPases (PMCAs) in the survival of SC neurons, in vitro. We also postulated that a decrease in PMCA2 expression could cause neuronal death during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The current studies were undertaken to define the specific contribution of PMCA2 to degeneration of SC neurons, the effectors downstream to PMCA2 mediating neuronal death and the triggers that reduce PMCA2 expression. We report that knockdown of PMCA2 in SC neurons decreases collapsin response mediator protein 1 (CRMP1) levels. This is followed by cell death. Silencing of CRMP1 expression also leads to neuronal loss. Kainic acid reduces both PMCA2 and CRMP1 levels and induces neuronal death. Administration of an alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist, at onset or peak of EAE, restores the decreased PMCA2 and CRMP1 levels to control values and ameliorates clinical deficits. Thus, our data link the reduction in PMCA2 expression with perturbations in the expression of CRMP1 and the ensuing death of SC neurons. This represents an additional mechanism underlying AMPA/kainate receptor-mediated excitotoxicity with relevance to neurodegeneration in EAE. PMID:20489728

  18. Hydrogen production by photosynthetic microorganisms

    SciTech Connect

    Akano, T.; Fukatsu, K.; Miyasaka, H. |

    1996-12-31

    Hydrogen is a clean energy alternative to the fossil fuels, the main source of greenhouse gas emissions. We developed a stable system for the conversion of solar energy into hydrogen using photosynthetic microorganisms. Our system consists of the following three stages: (1) Photosynthetic starch accumulation in green microalgae (400 L x2); (2) Dark anaerobic fermentation of the algal starch biomass to produce hydrogen and organic compounds (155 L x2); and (3) Further conversion of the organic compounds to produce hydrogen using photosynthetic bacteria (three types of reactors, parallel plate, raceway, and tubular). We constructed a test plant of this process at Nankoh power plant of Kansai Electric Power Company in Osaka, Japan, and carried out a series of tests using CO{sub 2} obtained from a chemical absorption pilot-plant. The photobiological hydrogen production process used a combination of a marine alga, Chlamydomonas sp. MGA 161 and marine photosynthetic bacterium, Rhodopseudomonas sp. W-1S. The dark anaerobic fermentation of algal starch biomass was also investigated. Sustained and stable starch accumulation, starch degradation in the algal cell, and hydrogen production from algal fermentation and photosynthetic bacteria in the light were demonstrated during several experiments. 3 refs., 12 figs., 1 tab.

  19. Acute cholesterol depletion impairs functional expression of tissue factor in fibroblasts: modulation of tissue factor activity by membrane cholesterol

    PubMed Central

    Mandal, Samir K.; Iakhiaev, Alexei; Pendurthi, Usha R.; Mohan Rao, L. Vijaya

    2010-01-01

    Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-β-cyclodextrin–reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding. PMID:15328160

  20. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

    SciTech Connect

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the Yxx{phi} domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1{sub NL4.3} compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and

  1. Functions of tocopherols in the cells of plants and other photosynthetic organisms.

    PubMed

    Mokrosnop, V M

    2014-01-01

    Tocopherol synthesis has only been observed in photosynthetic organisms (plants, algae and some cyanobacteria). Tocopherol is synthesized in the inner membrane of chloroplasts and distributed between chloroplast membranes, thylakoids and plastoglobules. Physiological significance of tocopherols for human and animal is well-studied, but relatively little is known about their function in plant organisms. Among the best characterized functions oftocopherols in cells is their ability to scavenge and quench reactive oxygen species and fat-soluble by-products of oxidative stress. There are the data on the participation of different mechanisms of α-tocopherol action in protecting photosystem II (PS II) from photoinhibition both by deactivation of singlet oxygen produced by PSII and by reduction of proton permeability of thylakoid membranes, leading to acidification of lumen under high light conditions and activation of violaxanthin de-epoxidase. Additional biological activity of tocopherols, independent of its antioxidant functions have been demonstrated. Basic mechanisms for these effects are connected with the modulation of signal transduction pathways by specific tocopherols and, in some instances, by transcriptional activation of gene expression. PMID:25816585

  2. Vesicle-associated membrane protein 7 is expressed in intestinal ER.

    PubMed

    Siddiqi, Shadab A; Mahan, James; Siddiqi, Shahzad; Gorelick, Fred S; Mansbach, Charles M

    2006-03-01

    Intestinal dietary triacylglycerol absorption is a multi-step process. Triacylglycerol exit from the endoplasmic reticulum (ER) is the rate-limiting step in the progress of the lipid from its apical absorption to its basolateral membrane export. Triacylglycerol is transported from the ER to the cis Golgi in a specialized vesicle, the pre-chylomicron transport vesicle (PCTV). The vesicle-associated membrane protein 7 (VAMP7) was found to be more concentrated on PCTVs compared with ER membranes. VAMP7 has been previously identified associated with post-Golgi sites in eukaryotes. To examine the potential role of VAMP7 in PCTV trafficking, antibodies were generated that identified a 25 kDa band consistent with VAMP7 but did not crossreact with VAMP1,2. VAMP7 was concentrated on intestinal ER by immunofluorescence microscopy. Immunoelectron microscopy showed that the ER proteins Sar1 and rBet1 were present on PCTVs and colocalized with VAMP7. Iodixanol gradient centrifugation showed VAMP7 to be isodense with ER and endosomes. Although VAMP7 localized to intestinal ER, it was not present in the ER of liver and kidney. Anti-VAMP7 antibodies reduced the transfer of triacylglycerol, but not newly synthesized proteins, from the ER to the Golgi by 85%. We conclude that VAMP7 is enriched in intestinal ER and that it plays a functional role in the delivery of triacylglycerol from the ER to the Golgi. PMID:16495485

  3. Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates

    PubMed Central

    Janot, Mathilde; Audfray, Aymeric; Loriol, Céline; Germot, Agnès; Maftah, Abderrahman; Dupuy, Fabrice

    2009-01-01

    Background Several global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes. Results Of the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries), providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation. Conclusion For the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis. PMID:19843320

  4. Arsenic biomethylation by photosynthetic organisms

    PubMed Central

    Ye, Jun; Rensing, Christopher; Rosen, Barry P.; Zhu, Yong-Guan

    2013-01-01

    Arsenic (As) is a ubiquitous element that is widespread in the environment and causes numerous health problems. Biomethylation of As has implications for its mobility and toxicity. Photosynthetic organisms may play a significant role in As geochemical cycling by methylating it to different As species, but little is known about the mechanisms of methylation. Methylated As species have been found in many photosynthetic organisms, and several arsenite S-adenosylmethionine (SAM) methyltransferases have been characterized in cyanobacteria and algae. However, higher plants may not have the ability to methylate As. Instead, methylated arsenicals in plants probably originate from microorganisms in soils and the rhizosphere. Here, we propose possible approaches for developing ‘smart’ photosynthetic organisms with an enhanced and sensitive biomethylation capacity for bioremediation and safer food. PMID:22257759

  5. Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli

    PubMed Central

    White, Jennifer; Lukacik, Petra; Esser, Dirk; Steward, Michael; Giddings, Naomi; Bright, Jeremy R.; Fritchley, Sarah J.; Morgan, B. Paul; Lea, Susan M.; Smith, Geoffrey P.; Smith, Richard A.G.

    2004-01-01

    Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential. PMID:15322283

  6. RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals.

    PubMed

    Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

    2016-05-01

    Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. PMID:27083698

  7. Three nuclear and two membrane estrogen receptors in basal teleosts, Anguilla sp.: Identification, evolutionary history and differential expression regulation.

    PubMed

    Lafont, Anne-Gaëlle; Rousseau, Karine; Tomkiewicz, Jonna; Dufour, Sylvie

    2016-09-01

    Estrogens interact with classical intracellular nuclear receptors (ESR), and with G-coupled membrane receptors (GPER). In the eel, we identified three nuclear (ESR1, ESR2a, ESR2b) and two membrane (GPERa, GPERb) estrogen receptors. Duplicated ESR2 and GPER were also retrieved in most extant teleosts. Phylogeny and synteny analyses suggest that they result from teleost whole genome duplication (3R). In contrast to conserved 3R-duplicated ESR2 and GPER, one of 3R-duplicated ESR1 has been lost shortly after teleost emergence. Quantitative PCRs revealed that the five receptors are all widely expressed in the eel, but with differential patterns of tissue expression and regulation. ESR1 only is consistently up-regulated in vivo in female eel BPG-liver axis during induced sexual maturation, and also up-regulated in vitro by estradiol in eel hepatocyte primary cultures. This first comparative study of the five teleost estradiol receptors provides bases for future investigations on differential roles that may have contributed to the conservation of multiple estrogen receptors. PMID:26654744

  8. Alterations of endothelial nucleotide levels by mycophenolic acid result in changes of membrane glycosylation and E-selectin expression.

    PubMed

    Bertalanffy, P; Dubsky, P; Wolner, E; Weigel, G

    1999-03-01

    The effect of the inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH), mycophenolic acid, on intracellular nucleotides and the synthesis of cellular glycoproteins was evaluated in human umbilical vein endothelial cells. A clinically attainable concentration (10 micromol/l) of mycophenolic acid decreased guanosine-5'-triphosphate (GTP) levels significantly and led to a strong elevation of uridine-5'-triphosphate (UTP), whereas intracellular adenosine-5'-triphosphate (ATP) pools remained unaffected. The staining of the endothelial cell membranes with lectins specific for fucose and mannose (Ulex europaeus- and Galanthus nivalis agglutinin, respectively) was reduced, reflecting an inhibition of fucose and mannose incorporation into endothelial glycoproteins. The surface expression of E-selectin, an important determinant for leuko-endothelial interactions decreased significantly. Guanine and guanosine prevented the actions of mycophenolic acid and reversed the drug-induced decrease in GTP and its associated effects. The findings that mycophenolic acid produces alterations in the formation of glycoproteins and in the membrane architecture are indicative of metabolic lesions induced by an agent that depresses guanine nucleotide synthesis through inhibition of IMPDH. The pronounced reduction of E-selectin surface expression on endothelial cells accompanied by changes of endothelial cell fucosylation, a prerequisite for the contact with lymphocytic L-selectin, indicates an inhibitory effect of mycophenolic acid in the rolling phase of leukocyte recruitment and strongly implies a new and additional immunosuppressive mechanism of this agent. PMID:10353469

  9. Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases

    PubMed Central

    Jaureguiberry, María S.; Tricerri, M. Alejandra; Sanchez, Susana A.; Finarelli, Gabriela S.; Montanaro, Mauro A.; Prieto, Eduardo D.; Rimoldi, Omar J.

    2014-01-01

    Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis. PMID:24473084

  10. RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals

    PubMed Central

    Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

    2016-01-01

    Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. PMID:27083698

  11. Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41

    PubMed Central

    Gong, Zhen; Kessans, Sarah A; Song, Lusheng; Dörner, Katerina; Lee, Ho-Hsien; Meador, Lydia R; LaBaer, Joshua; Hogue, Brenda G; Mor, Tsafrir S; Fromme, Petra

    2014-01-01

    The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR-TMTEV-6His was improved by fusion to the C-terminus of Mistic protein, yielding ∼1 mg of pure protein per liter. The isolated MPR-TMTEV-6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV-6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1. PMID:25155369

  12. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions.

    PubMed

    Azzi, Sandy; Gallerne, Cindy; Romei, Cristina; Le Coz, Vincent; Gangemi, Rosaria; Khawam, Krystel; Devocelle, Aurore; Gu, Yanhong; Bruno, Stefania; Ferrini, Silvano; Chouaib, Salem; Eid, Pierre; Azzarone, Bruno; Giron-Michel, Julien

    2015-06-01

    Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105(+)). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105(+), where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105(+) from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, "apparently normal" ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral "preneoplastic" environment committed to favor tumor progression. PMID:26152359

  13. Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

    PubMed Central

    Flick, Kirsten; Ahuja, Sanjay; Chene, Arnaud; Bejarano, Maria Teresa; Chen, Qijun

    2004-01-01

    Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons

  14. Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal non-overlapping epitopes.

    PubMed

    Feng, Yang; Wang, Yanping; Zhu, Zhongyu; Li, Wei; Sussman, Robyn T; Randall, Michael; Bosse, Kristopher R; Maris, John M; Dimitrov, Dimiter S

    2016-01-01

    CD56 (NCAM, neural cell adhesion molecule) is over-expressed in many tumor types, including neuroblastoma, multiple myeloma, small cell lung cancer, ovarian cancer, acute myeloid leukemia, NK-T lymphoma, neuroendocrine cancer and pancreatic cancer. Using phage display, we identified 2 high-affinity anti-CD56 human monoclonal antibodies (mAbs), m900 and m906, which bound to spatially separated non-overlapping epitopes with similar affinity (equilibrium dissociation constant 2.9 and 4.5 nM, respectively). m900 bound to the membrane proximal fibronectin type III-like domains, whereas m906 bound to the N-terminal IgG-like domains. m906 induced significant down-regulation of CD56 in 4 neuroblastoma cell lines tested, while m900-induced downregulation of CD56 was much lower. Antibody-drug conjugates (ADCs) made by conjugation with a highly potent pyrrolobenzodiazepine dimer (PBD) exhibited killing activity that correlated with CD56 down-regulation, and to some extent with in vivo binding ability of the antibodies. The m906PBD ADC was much more potent than m900PBD, likely due to higher CD56-mediated downregulation and stronger binding to cells. Treatment with m906PBD ADC resulted in very potent cytotoxicity (IC50: 0.05-1.7 pM). These results suggest a novel approach for targeting CD56-expressing neuroblastoma cells. Further studies in animal models and in humans are needed to find whether these antibodies and their drug conjugates are promising candidate therapeutics. PMID:26910291

  15. Thylakoid membrane function in heterocysts.

    PubMed

    Magnuson, Ann; Cardona, Tanai

    2016-03-01

    Multicellular cyanobacteria form different cell types in response to environmental stimuli. Under nitrogen limiting conditions a fraction of the vegetative cells in the filament differentiate into heterocysts. Heterocysts are specialized in atmospheric nitrogen fixation and differentiation involves drastic morphological changes on the cellular level, such as reorganization of the thylakoid membranes and differential expression of thylakoid membrane proteins. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase by developing an extra polysaccharide layer that limits air diffusion into the heterocyst and by upregulating heterocyst-specific respiratory enzymes. In this review article, we summarize what is known about the thylakoid membrane in heterocysts and compare its function with that of the vegetative cells. We emphasize the role of photosynthetic electron transport in providing the required amounts of ATP and reductants to the nitrogenase enzyme. In the light of recent high-throughput proteomic and transcriptomic data, as well as recently discovered electron transfer pathways in cyanobacteria, our aim is to broaden current views of the bioenergetics of heterocysts. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux. PMID:26545609

  16. Protection of the Photosynthetic Apparatus from Extreme Dehydration and Oxidative Stress in Seedlings of Transgenic Tobacco

    PubMed Central

    Personat, José-María; Tejedor-Cano, Javier; Lindahl, Marika; Diaz-Espejo, Antonio; Jordano, Juan

    2012-01-01

    A genetic program that in sunflower seeds is activated by Heat Shock transcription Factor A9 (HaHSFA9) has been analyzed in transgenic tobacco seedlings. The ectopic overexpression of the HSFA9 program protected photosynthetic membranes, which resisted extreme dehydration and oxidative stress conditions. In contrast, heat acclimation of seedlings induced thermotolerance but not resistance to the harsh stress conditions employed. The HSFA9 program was found to include the expression of plastidial small Heat Shock Proteins that accumulate only at lower abundance in heat-stressed vegetative organs. Photosystem II (PSII) maximum quantum yield was higher for transgenic seedlings than for non-transgenic seedlings, after either stress treatment. Furthermore, protection of both PSII and Photosystem I (PSI) membrane protein complexes was observed in the transgenic seedlings, leading to their survival after the stress treatments. It was also shown that the plastidial D1 protein, a labile component of the PSII reaction center, and the PSI core protein PsaB were shielded from oxidative damage and degradation. We infer that natural expression of the HSFA9 program during embryogenesis may protect seed pro-plastids from developmental desiccation. PMID:23227265

  17. GRP78-targeted nanotherapy against castrate-resistant prostate cancer cells expressing membrane GRP78.

    PubMed

    Delie, Florence; Petignat, Patrick; Cohen, Marie

    2013-12-01

    Glucose-regulated protein 78, GRP78, is a chaperone protein mainly located in the endoplasmic reticulum (ER) of normal cells. In stress conditions, GRP78 is overexpressed and in different cancer cell types, it is expressed at the cell surface, whereas it stays intracellular in non-cancerous cells. Therefore, it appears as a strategic target to recognize malignant cells. Prostate cancer is one of the most diagnosed cancers in men. The development of castrate resistant tumors and the resistance to chemotherapy frequently occur. The carboxy-terminal ER retention domain is defined by the KDEL amino acid sequence. We developed anti-KDEL functionalized polymeric nanoparticles (NPs) loaded with paclitaxel (Tx) to specifically target prostate cancer cells expressing GRP78. The sensitivity to Tx in different formulations was compared in three prostate cell lines: PNT1B, a normal cell line, PC3, a cancer cell line faintly expressing GRP78 at its surface, and DU145, a cancer cell line expressing GRP78 at its cell surface. Our results show that the targeted formulation significantly increases Tx sensitivity of cell line expressing GRP78 at its surface compared to other treatments suggesting the added value of GRP78 targeted therapy for castrate resistant tumor which expresses GRP78 at its cell surface. PMID:23090204

  18. Nobel lecture. The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis.

    PubMed Central

    Deisenhofer, J; Michel, H

    1989-01-01

    In our lectures we first describe the history and methods of membrane protein crystallization, before we show how the structure of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis was solved. Then the structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Finally we draw conclusions on the structure of the photosystem II reaction centre from plants and discuss the aspects of membrane protein structure. Sections 1 (crystallization), 4 (conclusions on the structure of photosystem II reaction centre and evolutionary aspects) and 5 (aspects of membrane protein structure) were presented and written by H.M., Sections 2 (determination of the structure) and 3 (structure and function) by J.D. We have arranged the paper in this way in order to facilitate continuous reading. Images PMID:2676514

  19. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    SciTech Connect

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P. )

    1991-02-15

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A){sup +} RNA.

  20. Photosynthetic reaction centers in bacteria

    SciTech Connect

    Norris, J.R. Univ. of Chicago, IL ); Schiffer, M. )

    1990-07-30

    The photochemistry of photosynthesis begins in complexes called reaction centers. These have become model systems to study the fundamental process by which plants and bacteria convert and store solar energy as chemical free energy. In green plants, photosynthesis occurs in two systems, each of which contains a different reaction center, working in series. In one, known as photosystem 1, oxidized nicotinamide adenine dinucleotide phosphate (NADP[sup +]) is reduced to NADPH for use in a series of dark reactions called the Calvin cycle, named for Nobel Laureate Melvin Calvin, by which carbon dioxide is converted into useful fuels such as carbohydrates and sugars. In the other half of the photosynthetic machinery of green plants, called photosystem 2, water is oxidized to produce molecular oxygen. A different form of photosynthesis occurs in photosynthetic bacteria, which typically live at the bottom of ponds and feed on organic debris. Two main types of photosynthetic bacteria exist: purple and green. Neither type liberates oxygen from water. Instead, the bacteria feed on organic media or inorganic materials, such as sulfides, which are easier to reduce or oxidize than carbon dioxide or water. Perhaps in consequence, their photosynthetic machinery is simpler than that of green, oxygen-evolving plants and their primary photochemistry is better understood.

  1. Spectral measurements of photosynthetic efficiency

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The photosynthetic efficiency of plants was examined for plants in two very different canopies, a USDA cornfield having an instrumented flux tower in Beltsville, MD, USA and a coniferous forest in British Columbia, Canada, included in the tower network of the Canadian Carbon Program. Basic field st...

  2. The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii.

    PubMed

    Juergens, Matthew T; Deshpande, Rahul R; Lucker, Ben F; Park, Jeong-Jin; Wang, Hongxia; Gargouri, Mahmoud; Holguin, F Omar; Disbrow, Bradley; Schaub, Tanner; Skepper, Jeremy N; Kramer, David M; Gang, David R; Hicks, Leslie M; Shachar-Hill, Yair

    2015-02-01

    The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and (13)C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700(+) reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic (13)CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates. PMID:25489023

  3. The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii1[OPEN

    PubMed Central

    Juergens, Matthew T.; Deshpande, Rahul R.; Lucker, Ben F.; Park, Jeong-Jin; Wang, Hongxia; Gargouri, Mahmoud; Holguin, F. Omar; Disbrow, Bradley; Schaub, Tanner; Skepper, Jeremy N.; Kramer, David M.; Gang, David R.; Hicks, Leslie M.; Shachar-Hill, Yair

    2015-01-01

    The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and 13C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700+ reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic 13CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates. PMID:25489023

  4. Phosphofructokinase muscle-specific isoform requires caveolin-3 expression for plasma membrane recruitment and caveolar targeting: implications for the pathogenesis of caveolin-related muscle diseases.

    PubMed

    Sotgia, Federica; Bonuccelli, Gloria; Minetti, Carlo; Woodman, Scott E; Capozza, Franco; Kemp, Robert G; Scherer, Philipp E; Lisanti, Michael P

    2003-12-01

    Previous co-immunoprecipitation studies have shown that endogenous PFK-M (phosphofructokinase, muscle-specific isoform) associates with caveolin (Cav)-3 under certain metabolic conditions. However, it remains unknown whether Cav-3 expression is required for the plasma membrane recruitment and caveolar targeting of PFK-M. Here, we demonstrate that recombinant expression of Cav-3 dramatically affects the subcellular localization of PFK-M, by targeting PFK-M to the plasma membrane, and by trans-locating PFK-M to caveolae-enriched membrane domains. In addition, we show that the membrane recruitment and caveolar targeting of PFK-M appears to be strictly dependent on the concentration of extracellular glucose. Interestingly, recombinant expression of PFK-M with three Cav-3 mutants [DeltaTFT (63 to 65), P104L, and R26Q], which harbor the same mutations as seen in the human patients with Cav-3-related muscle diseases, causes a substantial reduction in PFK-M expression levels, and impedes the membrane recruitment of PFK-M. Analysis of skeletal muscle tissue samples from Cav-3(-/-) mice directly demonstrates that Cav-3 expression regulates the phenotypic behavior of PFK-M. More specifically, in Cav-3-null mice, PFK-M is no longer targeted to the plasma membrane, and is excluded from caveolar membrane domains. As such, our current results may be important in understanding the pathogenesis of Cav-3-related muscle diseases, such as limb-girdle muscular dystrophy-1C, distal myopathy, and rippling muscle disease, that are caused by mutations within the human Cav-3 gene. PMID:14633633

  5. Plasma Membrane –Cell Wall Adhesion Is Required for Expression of Plant Defense Responses during Fungal Penetration

    PubMed Central

    Mellersh, Denny G.; Heath, Michèle C.

    2001-01-01

    Fungal pathogens almost invariably trigger cell wall–associated defense responses, such as extracellular hydrogen peroxide generation and callose deposition, when they attempt to penetrate either resistant or susceptible plant cells. In the current study, we provide evidence that the expression of these defenses is dependent on adhesion between the plant cell wall and the plasma membrane. Peptides containing an Arg-Gly-Asp (RGD) motif, which interfered with plasma membrane–cell wall adhesion as shown by the loss of the thin plasma membrane–cell wall connections known as Hechtian strands, reduced the expression of cell wall–associated defense responses during the penetration of nonhost plants by biotrophic fungal pathogens. This reduction was associated with increased fungal penetration efficiency. Neither of these effects was seen after treatment with similar peptides lacking the RGD motif. Disruption of plant microfilaments had no effect on Hechtian strands but mimicked the effect of RGD peptides on wall defenses, suggesting that the expression of cell wall–associated defenses involves communication between the plant cell wall and the cytosol across the plasma membrane. To visualize the state of the plasma membrane–cell wall interaction during fungal penetration, we observed living cells during sucrose-induced plasmolysis. In interactions that were characterized by the early expression of cell wall–associated defenses, there was no change, or an increase, in plasma membrane–cell wall adhesion under the penetration point as the fungus grew through the plant cell wall. In contrast, for rust fungus interactions with host plants, there was a strong correlation between a lack of cell wall–associated defenses and a localized decrease in plasma membrane–cell wall adhesion under the penetration point. Abolition of this localized decreased adhesion by previous inoculation with a fungus that increased plasma membrane–cell wall adhesion resulted in

  6. Effects of Unloading and Reloading on Expressions of Skelatal Muscle Membrane Proteins in Mice

    NASA Astrophysics Data System (ADS)

    Ohno, Y.; Ikuta, A.; Goto, A.; Sugiura, T.; Ohira, Y.; Yoshioka, T.; Goto, K.

    2013-02-01

    Effects of unloading and reloading on the expression levels of tripartite motif-containing 72 (TRIM72) and caveolin-3 (Cav-3) of soleus muscle in mice were investigated. Male C57BL/6J mice (11-week old) were randomly assigned to control and hindlimb-suspended groups. Some of mice in hindlimb-suspended group were subjected to continuous hindlimb suspension (HS) for 2 weeks with or without 7 days of ambulation recovery. Following HS, the muscle weight and protein expression levels of TRIM72 and Cav-3 in soleus were decreased. On the other hand, the gradual increases in muscle mass, TRIM72 and Cav-3 were observed after reloading following HS. Therefore, it was suggested that mechanical loading played a key role in a regulatory system for protein expressions of TRIM72 and Cav-3.

  7. Matrigel Basement Membrane Matrix influences expression of microRNAs in cancer cell lines

    SciTech Connect

    Price, Karina J.; Tsykin, Anna; Giles, Keith M.; Sladic, Rosemary T.; Epis, Michael R.; Ganss, Ruth; Goodall, Gregory J.; Leedman, Peter J.

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Matrigel alters cancer cell line miRNA expression relative to culture on plastic. Black-Right-Pointing-Pointer Many identified Matrigel-regulated miRNAs are implicated in cancer. Black-Right-Pointing-Pointer miR-1290, -210, -32 and -29b represent a Matrigel-induced miRNA signature. Black-Right-Pointing-Pointer miR-32 down-regulates Integrin alpha 5 (ITGA5) mRNA. -- Abstract: Matrigel is a medium rich in extracellular matrix (ECM) components used for three-dimensional cell culture and is known to alter cellular phenotypes and gene expression. microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and have roles in cancer. While miRNA profiles of numerous cell lines cultured on plastic have been reported, the influence of Matrigel-based culture on cancer cell miRNA expression is largely unknown. This study investigated the influence of Matrigel on the expression of miRNAs that might facilitate ECM-associated cancer cell growth. We performed miRNA profiling by microarray using two colon cancer cell lines (SW480 and SW620), identifying significant differential expression of miRNAs between cells cultured in Matrigel and on plastic. Many of these miRNAs have previously been implicated in cancer-related processes. A common Matrigel-induced miRNA signature comprised of up-regulated miR-1290 and miR-210 and down-regulated miR-29b and miR-32 was identified using RT-qPCR across five epithelial cancer cell lines (SW480, SW620, HT-29, A549 and MDA-MB-231). Experimental modulation of these miRNAs altered expression of their known target mRNAs involved in cell adhesion, proliferation and invasion, in colon cancer cell lines. Furthermore, ITGA5 was identified as a novel putative target of miR-32 that may facilitate cancer cell interactions with the ECM. We propose that culture of cancer cell lines in Matrigel more accurately recapitulates miRNA expression and function in cancer than culture on plastic and thus is a

  8. Expression of the Domain Cassette 8 Plasmodium falciparum Erythrocyte Membrane Protein 1 Is Associated with Cerebral Malaria in Benin

    PubMed Central

    Bertin, Gwladys I.; Lavstsen, Thomas; Guillonneau, François; Doritchamou, Justin; Wang, Christian W.; Jespersen, Jakob S.; Ezimegnon, Sem; Fievet, Nadine; Alao, Maroufou J.; Lalya, Francis; Massougbodji, Achille; Ndam, Nicaise Tuikue; Theander, Thor G.; Deloron, Philippe

    2013-01-01

    Background Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi-conserved types defined by tandem runs of specific domains (“domain cassettes” (DC)). The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection. Methods Parasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM). We assessed the transcript level of var genes by RT-qPCR and the expression of PfEMP-1 proteins by LC-MS/MS. Results Var genes encoding DC8 and Group A PfEMP-1 were transcribed more often and at higher levels in cerebral malaria vs. uncomplicated malaria patients. LC-MS/MS identified peptides from group A, DC8 PfEMP-1 more frequently in cerebral malaria than in uncomplicated malaria and pregnancy-associated malaria samples. Conclusion This is the first study to show association between PfEMP-1 subtype and disease outcome by direct analysis of parasites proteome. The results corroborate that group A and specifically the PfEMP-1 types DC8 are universally associated with cerebral malaria. This is a crucial observation for promoting studies on malaria pathogenesis. PMID:23922654

  9. A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic.

    PubMed Central

    Murdin, A D; Su, H; Manning, D S; Klein, M H; Parnell, M J; Caldwell, H D

    1993-01-01

    Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines. Images PMID:7691749

  10. Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro

    PubMed Central

    Saw, Valerie P.J.; Offiah, Ifeoma; Dart, Robin J.; Galatowicz, Grazyna; Dart, John K.G.; Daniels, Julie T.; Calder, Virginia L.

    2009-01-01

    Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts. PMID:19910508

  11. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

    SciTech Connect

    Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

    2006-04-01

    The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials.

  12. The membrane skeleton in Paramecium: Molecular characterization of a novel epiplasmin family and preliminary GFP expression results.

    PubMed

    Pomel, Sébastien; Diogon, Marie; Bouchard, Philippe; Pradel, Lydie; Ravet, Viviane; Coffe, Gérard; Viguès, Bernard

    2006-02-01

    Previous attempts to identify the membrane skeleton of Paramecium cells have revealed a protein pattern that is both complex and specific. The most prominent structural elements, epiplasmic scales, are centered around ciliary units and are closely apposed to the cytoplasmic side of the inner alveolar membrane. We sought to characterize epiplasmic scale proteins (epiplasmins) at the molecular level. PCR approaches enabled the cloning and sequencing of two closely related genes by amplifications of sequences from a macronuclear genomic library. Using these two genes (EPI-1 and EPI-2), we have contributed to the annotation of the Paramecium tetraurelia macronuclear genome and identified 39 additional (paralogous) sequences. Two orthologous sequences were found in the Tetrahymena thermophila genome. Structural analysis of the 43 sequences indicates that the hallmark of this new multigenic family is a 79 aa domain flanked by two Q-, P- and V-rich stretches of sequence that are much more variable in amino-acid composition. Such features clearly distinguish members of the multigenic family from epiplasmic proteins previously sequenced in other ciliates. The expression of Green Fluorescent Protein (GFP)-tagged epiplasmin showed significant labeling of epiplasmic scales as well as oral structures. We expect that the GFP construct described herein will prove to be a useful tool for comparative subcellular localization of different putative epiplasmins in Paramecium. PMID:16427359

  13. Expression of a mitochondrial progesterone receptor in human spermatozoa correlates with a progestin-dependent increase in mitochondrial membrane potential.

    PubMed

    Tantibhedhyangkul, J; Hawkins, K C; Dai, Q; Mu, K; Dunn, C N; Miller, S E; Price, T M

    2014-11-01

    The hyperactivation of human spermatozoa necessary for fertilization requires a substantial increase in cellular energy production. The factors responsible for increasing cellular energy remain poorly defined. This article proposes a role for a novel mitochondrial progesterone receptor (PR-M) in modulation of mitochondrial activity. Basic science studies demonstrate a 38 kDa protein with western blot analysis, consistent with PR-M; whereas imaging studies with confocal and immunoelectron microscopy demonstrate a PR on the mitochondria. Treatment with a PR-specific progestin shows increased mitochondrial membrane potential, not related to induction of an acrosome reaction. The increase in mitochondrial membrane potential was inhibited by a specific PR antagonist, but not affected by an inhibitor to the progesterone-dependent Catsper voltage-activated channel. In conclusion, these studies suggest expression of a novel mitochondrial PR in human spermatozoa with a progestin-dependent increase in mitochondrial activity. This mechanism may serve to enhance cellular energy production as the spermatozoa traverse the female genital tract being exposed to increasing concentrations of progesterone. PMID:25187426

  14. Quantum oscillatory exciton migration in photosynthetic reaction centers

    NASA Astrophysics Data System (ADS)

    Abramavicius, Darius; Mukamel, Shaul

    2010-08-01

    The harvesting of solar energy and its conversion to chemical energy is essential for all forms of life. The primary photon absorption, transport, and charge separation events, which trigger a chain of chemical reactions, take place in membrane-bound photosynthetic complexes. Whether quantum effects, stemming from entanglement of chromophores, persist in the energy transport at room temperature, despite the rapid decoherence effects caused by environment fluctuations, is under current active debate. If confirmed, these may explain the high efficiency of light harvesting and open up numerous applications to quantum computing and information processing. We present simulations of the photosynthetic reaction center of photosystem II that clearly establish oscillatory energy transport at room temperature originating from interference of quantum pathways. These signatures of quantum transport may be observed by two dimensional coherent optical spectroscopy.

  15. Influence of thermal light correlations on photosynthetic structures

    NASA Astrophysics Data System (ADS)

    de Mendoza, Adriana; Manrique, Pedro; Caycedo-Soler, Felipe; Johnson, Neil F.; Rodríguez, Ferney J.; Quiroga, Luis

    2014-03-01

    The thermal light from the sun is characterized by both classical and quantum mechanical correlations. These correlations have left a fingerprint on the natural harvesting structures developed through five billion years of evolutionary pressure, specially in photosynthetic organisms. In this work, based upon previous extensive studies of spatio-temporal correlations of light fields, we hypothesize that structures involving photosensitive pigments like those present in purple bacteria vesicles emerge as an evolutionary response to the different properties of incident light. By using burstiness and memory as measures that quantify higher moments of the photon arrival statistics, we generate photon-time traces. They are used to simulate absorption on detectors spatially extended over regions comparable to these light fields coherence length. Finally, we provide some insights into the connection between these photo-statistical features with the photosynthetic membrane architecture and the lights' spatial correlation. Facultad de Ciencias Uniandes.

  16. Ion antiport accelerates photosynthetic acclimation in fluctuating light environments

    PubMed Central

    Armbruster, Ute; Carrillo, L. Ruby; Venema, Kees; Pavlovic, Lazar; Schmidtmann, Elisabeth; Kornfeld, Ari; Jahns, Peter; Berry, Joseph A.; Kramer, David M.; Jonikas, Martin C.

    2014-01-01

    Many photosynthetic organisms globally, including crops, forests and algae, must grow in environments where the availability of light energy fluctuates dramatically. How photosynthesis maintains high efficiency despite such fluctuations in its energy source remains poorly understood. Here we show that Arabidopsis thaliana K+ efflux antiporter (KEA3) is critical for high photosynthetic efficiency under fluctuating light. On a shift from dark to low light, or high to low light, kea3 mutants show prolonged dissipation of absorbed light energy as heat. KEA3 localizes to the thylakoid membrane, and allows proton efflux from the thylakoid lumen by proton/potassium antiport. KEA3’s activity accelerates the downregulation of pH-dependent energy dissipation after transitions to low light, leading to faster recovery of high photosystem II quantum efficiency and increased CO2 assimilation. Our results reveal a mechanism that increases the efficiency of photosynthesis under fluctuating light. PMID:25451040

  17. Ion antiport accelerates photosynthetic acclimation in fluctuating light environments.

    PubMed

    Armbruster, Ute; Carrillo, L Ruby; Venema, Kees; Pavlovic, Lazar; Schmidtmann, Elisabeth; Kornfeld, Ari; Jahns, Peter; Berry, Joseph A; Kramer, David M; Jonikas, Martin C

    2014-01-01

    Many photosynthetic organisms globally, including crops, forests and algae, must grow in environments where the availability of light energy fluctuates dramatically. How photosynthesis maintains high efficiency despite such fluctuations in its energy source remains poorly understood. Here we show that Arabidopsis thaliana K(+) efflux antiporter (KEA3) is critical for high photosynthetic efficiency under fluctuating light. On a shift from dark to low light, or high to low light, kea3 mutants show prolonged dissipation of absorbed light energy as heat. KEA3 localizes to the thylakoid membrane, and allows proton efflux from the thylakoid lumen by proton/potassium antiport. KEA3's activity accelerates the downregulation of pH-dependent energy dissipation after transitions to low light, leading to faster recovery of high photosystem II quantum efficiency and increased CO2 assimilation. Our results reveal a mechanism that increases the efficiency of photosynthesis under fluctuating light. PMID:25451040

  18. Pigment oligomers as natural and artificial photosynthetic antennas

    SciTech Connect

    Blankenship, R.E.

    1996-12-31

    Green photosynthetic bacteria contain antenna complexes known as chlorosomes. These complexes are appressed to the cytoplasmic side of the inner cell membrane and function to absorb light and transfer the energy to the photochemical reaction center, where photochemical energy storage takes place. Chlorosomes differ from all other known photosynthetic antenna complexes in that the geometrical arrangement of pigments is determined primarily by pigment-pigment interactions instead of pigment-protein interactions. The bacteriochlorophyll c, d or e pigments found in chlorosomes form large oligomers with characteristic spectral properties significantly perturbed from those exhibited by monomeric pigments. Because of their close spatial interaction, the pigments are thought to be strongly coupled electronically, and many of the optical properties result from exciton interactions. This presentation will summarize existing knowledge on the chemical composition and properties of chlorosomes, the evidence for the oligomeric nature of chlorosome pigment organization and proposed structures for the oligomers, and the kinetics and mechanisms of energy transfer in chlorosomes.

  19. Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB

    SciTech Connect

    Tanabe, Mikio; Iverson, Tina M.

    2010-01-28

    The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P6{sub 3}. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P6{sub 3} crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P6{sub 3} crystal form had the best diffraction quality, yielding data extending to 2.3 {angstrom} resolution.

  20. Temporal expression of a membrane-associated protein putatively involved in repression of initiation of DNA replication in Bacillus subtilis.

    PubMed Central

    Eident-Wilkinson, B; Mele, L; Laffan, J; Firshein, W

    1992-01-01

    A Bacillus subtilis membrane-associated protein that binds specifically to the origin region of DNA replication may act as an inhibitor of DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). This protein, originally estimated to be 64 kDa, had a slightly lower molecular size (57 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during these studies. The size difference may be due to processing that results in modification of the protein. The protein can be extracted from both cytosol and membrane fractions, and the amounts in these fractions vary during the developmental cycle of B. subtilis. A complex pattern of expression in which significant levels were detected in spores was revealed; levels decreased dramatically during germination and increased after the first round of DNA replication. The decrease during germination was due to protease activity, as demonstrated by the addition of protease inhibitors and radioactive-labeling chase experiments. During vegetative growth, the protein levels increased until stationary phase, after which there was another decrease during sporulation. The decrease during sporulation may be partially due to sequestering of the protein into forespores, since as the putative repressor protein decreased in the mother cell, it increased in the forespores. However, protease activity was also involved in the decrease in the mother cell. The changes in expression of this protein are consistent with its role as a repressor of initiation of DNA replication. Additional studies, including sequence analysis and further antibody analysis, show that this protein is not a subunit of the pyruvate dehydrogenase complex. This relationship had been a possibility based upon the results of others (H. Hemila, A. Pavla, L. Paulin, S. Arvidson, and I. Palva, J. Bacteriol. 172:5052-5063, 1990). Images PMID:1729239

  1. A novel meningococcal outer membrane vesicle vaccine with constitutive expression of FetA: A phase I clinical trial

    PubMed Central

    Marsay, L.; Dold, C.; Green, C.A.; Rollier, C.S.; Norheim, G.; Sadarangani, M.; Shanyinde, M.; Brehony, C.; Thompson, A.J.; Sanders, H.; Chan, H.; Haworth, K.; Derrick, J.P.; Feavers, I.M.; Maiden, M.C.; Pollard, A.J.

    2015-01-01

    Summary Objectives Outer membrane vesicle (OMV) vaccines are used against outbreaks of capsular group B Neisseria meningitidis (MenB) caused by strains expressing particular PorA outer membrane proteins (OMPs). Ferric enterobactin receptor (FetA) is another variable OMP that induces type-specific bactericidal antibodies, and the combination of judiciously chosen PorA and FetA variants in vaccine formulations is a potential approach to broaden protection of such vaccines. Methods The OMV vaccine MenPF-1 was generated by genetically modifying N. meningitidis strain 44/76 to constitutively express FetA. Three doses of 25 μg or 50 μg of MenPF-1 were delivered intra-muscularly to 52 healthy adults. Results MenPF-1 was safe and well tolerated. Immunogenicity was measured by serum bactericidal assay (SBA) against wild-type and isogenic mutant strains. After 3 doses, the proportion of volunteers with SBA titres ≥1:4 (the putative protective titre) was 98% for the wild-type strain, and 77% for the strain 44/76 FetAonPorAoff compared to 51% in the strain 44/76 FetAoffPorAoff, demonstrating that vaccination with MenPF-1 simultaneously induced FetA and PorA bactericidal antibodies. Conclusion This study provides a proof-of-concept for generating bactericidal antibodies against FetA after OMV vaccination in humans. Prevalence-based choice of PorA and FetA types can be used to formulate a vaccine for broad protection against MenB disease. PMID:25982025

  2. Extracellular and Luminal pH Regulation by Vacuolar H+-ATPase Isoform Expression and Targeting to the Plasma Membrane and Endosomes*

    PubMed Central

    Smith, Gina A.; Howell, Gareth J.; Phillips, Clair; Muench, Stephen P.; Ponnambalam, Sreenivasan; Harrison, Michael A.

    2016-01-01

    Plasma membrane vacuolar H+-ATPase (V-ATPase) activity of tumor cells is a major factor in control of cytoplasmic and extracellular pH and metastatic potential, but the isoforms involved and the factors governing plasma membrane recruitment remain uncertain. Here, we examined expression, distribution, and activity of V-ATPase isoforms in invasive prostate adenocarcinoma (PC-3) cells. Isoforms 1 and 3 were the most highly expressed forms of membrane subunit a, with a1 and a3 the dominant plasma membrane isoforms. Correlation between plasma membrane V-ATPase activity and invasiveness was limited, but RNAi knockdown of either a isoform did slow cell proliferation and inhibit invasion in vitro. Isoform a1 was recruited to the cell surface from the early endosome-recycling complex pathway, its knockdown arresting transferrin receptor recycling. Isoform a3 was associated with the late endosomal/lysosomal compartment. Both a isoforms associated with accessory protein Ac45, knockdown of which stalled transit of a1 and transferrin-transferrin receptor, decreased proton efflux, and reduced cell growth and invasiveness; this latter effect was at least partly due to decreased delivery of the membrane-bound matrix metalloproteinase MMP-14 to the plasma membrane. These data indicate that in prostatic carcinoma cells, a1 and a3 isoform populations predominate in different compartments where they maintain different luminal pH. Ac45 plays a central role in navigating the V-ATPase to the plasma membrane, and hence it is an important factor in expression of the invasive phenotype. PMID:26912656

  3. Surface membrane antigen expression changes induced in vitro by exogenous growth factors in chronic lymphocytic leukemia cells.

    PubMed

    Vilpo, J; Hulkkonen, J; Hurme, M; Vilpo, L

    2002-09-01

    The factors determining the growth and survival of cells in B chronic lymphocytic leukemia (CLL) have remained poorly understood. We investigated the effects of optimal mitogen combinations (OMCs) on the expression of 26 surface membrane antigens among 33 CLL patients. The seven OMCs used were selected after pre-testing 14 combinations of (1) S. aureus Cowan I (SAC), (2) interleukin-2 (IL-2), (3) tumor necrosis factor alpha (TNF-alpha) and (4) 12-O-tetradecanoylphorbol 13-acetate (TPA; also known as phorbol 12-myristate 13-acetate or PMA). In flow cytometry we revealed that OMCs induced statistically highly significant upregulation of the expression of CD5, CD11c, CD19, CD22, CD23, CD25, CD38, CD40, CD45, CD45RO, CD95, CD126, CD130 and FMC7, and downregulation of CD20 and CD124 expression. Interestingly, the expression of CD27, CD45RA, CD79b, CD80, CD122 and that of the immunoglobulin gene superfamily members CD21, Ig-kappa, Ig-lambda, Ig-delta and Ig-micro were not significantly affected under similar conditions. The expression of several antigens was co-regulated, suggesting common regulatory pathways. These antigens include CD11c/CD5, CD11c/CD22, CD11c/CD126, CD11c/FMC7 as well as CD27/CD45, CD27/CD45RA and CD27/CD79b. Upregulation of surface antigen expression, induced by OMCs, should be applicable in antibody therapy in vitro and in vivo, and in negative stem cell selection for autotransplantation. Furthermore, the current strategy to enhance cell surface antigen expression may be a versatile tool to raise humoral and cell-mediated host defense against CLL cells. Upregulation of proteins mediating positive growth signals (eg CD25, CD40) and negative signals or apoptosis (eg CD95) may be used to sensitize cells to chemotherapy and programmed cell death. PMID:12200683

  4. The significance of the increased expression of phosphorylated MeCP2 in the membranes from patients with proliferative diabetic retinopathy.

    PubMed

    Li, Xiaohua; Liu, Xiaohui; Guo, Haoyi; Zhao, Zhaoxia; Li, Yun Sui; Chen, Guoming

    2016-01-01

    The purpose of this study was to evaluate the correlation of expression of phosphorylated methyl-CpG binding protein 2-Ser421 (MeCP2-S421) and VEGF in the membranes of patients with PDR. We examined the expression of phospho-MeCP2-S80, S421, VEGF and PEDF in surgically excised PDR membranes from 33 patients with diabetes, and idiopathic epiretinal membranes from 11 patients without diabetes, using immunohistochemistry and western blot. The colocalization of MeCP2-S421 with VEGF, PEDF, CD31, GFAP and αSMA was revealed by fluorescent double labeling. The effect of CoCl2 and knock down MeCP2 using specific siRNA on the expression of MeCP2 and VEGF were analyzed in HUCAC cells by Western blot. We found that phospho-MeCP2-S421 was significantly increased in the membranes from the patients with PDR compared with the specimens from patients without diabetes (P < 0.01). The expression of phospho-MeCP2-S421 was much stronger than that of phospho-MeCP2-S80 in the PDR membranes. Double labeling showed that the high phospho-MeCP2-S421 expression was associated with strong expression of VEGF, but not PEDF. Further, phospho-MeCP2-S421 and VEGF were increased by the stimulation of CoCl2 and knock down MeCP2 inhibited the expression of VEGF. Our result suggests that phospho-MeCP2-S421 might involve in the pathogenesis of PDR. PMID:27616658

  5. Fluorescent probe for high-throughput screening of membrane protein expression

    PubMed Central

    Backmark, A E; Olivier, N; Snijder, A; Gordon, E; Dekker, N; Ferguson, A D

    2013-01-01

    Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture. PMID:23776061

  6. Expression of the γ-Aminobutyric Acid (GABA) Plasma Membrane Transporter-1 in Monkey and Human Retina

    PubMed Central

    Casini, Giovanni; Rickman, Dennis W.; Brecha, Nicholas C.

    2010-01-01

    Purpose To determine the expression pattern of the predominant γ-aminobutyric acid (GABA) plasma membrane transporter GAT-1 in Old World monkey (Macaca mulatta) and human retina. Methods GAT-1 was localized in retinal sections by using immunohistochemical techniques with fluorescence and confocal microscopy. Double-labeling studies were performed with the GAT-1 antibody using antibodies to GABA, vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH), and the bipolar cell marker Mab115A10. Results The pattern of GAT-1 immunostaining was similar in human and monkey retinas. Numerous small immunoreactive somata were in the inner nuclear layer (INL) and were present rarely in the inner plexiform layer (IPL) of all retinal regions. Medium GAT-1 somata were in the ganglion cell layer in the parafoveal and peripheral retinal regions. GAT-1 fibers were densely distributed throughout the IPL. Varicose processes, originating from both the IPL and somata in the INL, arborized in the outer plexiform layer (OPL), forming a sparse network in all retinal regions, except the fovea. Sparsely occurring GAT-1 processes were in the nerve fiber layer in parafoveal regions and near the optic nerve head but not in the optic nerve. In the INL, 99% of the GAT-1 somata contained GABA, and 66% of the GABA immunoreactive somata expressed GAT-1. GAT-1 immunoreactivity was in all VIP-containing cells, but it was absent in TH-immunoreactive amacrine cells and in Mab115A10 immunoreactive bipolar cells. Conclusions GAT-1 in primate retinas is expressed by amacrine and displaced amacrine cells. The predominant expression of GAT-1 in the inner retina is consistent with the idea that GABA transporters influence neurotransmission and thus participate in visual information processing in the retina. PMID:16565409

  7. Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

    PubMed

    McShane, Adam J; Bajrami, Bekim; Ramos, Alex A; Diego-Limpin, Pamela A; Farrokhi, Vahid; Coutermarsh, Bonita A; Stanton, Bruce A; Jensen, Tim; Riordan, John R; Wetmore, Diana; Joseloff, Elizabeth; Yao, Xudong

    2014-11-01

    Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel's function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy. PMID:25227318

  8. Targeted Proteomic Quantitation of the Absolute Expression and Turnover of Cystic Fibrosis Transmembrane Conductance Regulator in the Apical Plasma Membrane

    PubMed Central

    2015-01-01

    Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel’s function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy. PMID:25227318

  9. Human carcinomas variably express the complement inhibitory proteins CD46 (membrane cofactor protein), CD55 (decay-accelerating factor), and CD59 (protectin).

    PubMed Central

    Niehans, G. A.; Cherwitz, D. L.; Staley, N. A.; Knapp, D. J.; Dalmasso, A. P.

    1996-01-01

    Normal human tissues express membrane-associated complement inhibitory proteins that protect these tissues from damage by autologous complement. To determine whether neoplasms also express these proteins, we examined the distribution of the complement inhibitors decay-accelerating factor (DAF), CD59 (protectin), and membrane cofactor protein in frozen samples of human breast, colon, kidney, and lung carcinomas and in adjacent non-neoplastic tissues, using immunohistochemistry. All samples were also studied for deposition of C3 fragments and activated C5b-9. Differences between normal tissues and the corresponding neoplasms were often observed, with loss or gain of expression of one or more inhibitors. Ductal carcinomas of the breast showed the most variation in phenotype; some tumors expressed only one inhibitor while others expressed different combinations of two or three inhibitors. Colon carcinomas, by contrast, stained intensely for all inhibitors. Renal cell carcinomas had weak to moderate expression of one to three inhibitors, generally DAF and CD59, whereas non-small cell carcinomas of the lung usually expressed CD59 and membrane cofactor protein with variable DAF immunoreactivity. The two small cell carcinomas of the lung showed little or no staining for any inhibitor. Activated C5b-9 deposition was seen adjacent to tumor nests in a minority of carcinomas and showed no correlation with complement inhibitor expression. C3 fragment deposition was minimal. Our results demonstrate that most carcinomas, with the exception of small cell carcinomas of the lung, do express one or more complement inhibitors at a level likely to inhibit complement-mediated cellular damage. Unexpectedly, large quantities of DAF and CD59 were often observed in tumor stroma, with only limited deposition in normal connective tissue. This suggests that carcinomas may supplement the activity of membrane-associated complement inhibitors by release of soluble forms of DAF and CD59 into the

  10. Lipopolysaccharide induces nitric oxide synthase expression and platelet-activating factor increases nitric oxide production in human fetal membranes in culture

    PubMed Central

    Seyffarth, Gunter; Nelson, Paul N; Dunmore, Simon J; Rodrigo, Nalinda; Murphy, Damian J; Carson, Ray J

    2004-01-01

    Background Platelet-activating factor and nitric oxide may be involved in the initiation of human labour as inflammatory mediators. The aim of this study was to test whether platelet-activating factor and lipopolysaccharide were able to induce nitric oxide synthase expression and stimulate the production of nitric oxide in human fetal membrane explants in culture. Methods Fetal membranes were collected from Caesarean sections at term. RNA was extracted from membranes and subjected to a qualitative RT-PCR to assess the baseline expression of iNOS. Discs of fetal membranes were cultured for 24 hours in the presence of platelet-activating factor at a dose range of 0.1 nanomolar – 1 micomolar or 1 microgram/ml lipopolysaccharide. Nitric oxide production was measured via nitrite ions in the culture medium and mRNA for iNOS was detected by RT-PCR. Results Culturing the membrane discs in medium containing serum induced nitric oxide synthase expression and platelet-activating factor significantly stimulated the production of nitric oxide under these conditions. When cultured without serum inducible nitric oxide synthase expression was induced by lipopolysaccharide, but not by platelet-activating factor. Conclusion Platelet-activating factor may have a role in the initiation of labour, at term or preterm, via the increased local production of nitric oxide as an inflammatory mediator. In this model of intrauterine infection, lipopolysaccharide was found to induce iNOS expression by fetal membranes, and this mechanism could be involved in preterm labour. PMID:15191613

  11. Phytochromes in photosynthetically competent plants

    SciTech Connect

    Pratt, L.H.

    1990-07-01

    Plants utilize light as a source of information in photomorphogenesis and of free energy in photosynthesis, two processes that are interrelated in that the former serves to increase the efficiency with which plants can perform the latter. Only one pigment involved in photomorphogenesis has been identified unequivocally, namely phytochrome. The thrust of this proposal is to investigate this pigment and its mode(s) of action in photosynthetically competent plants. Our long term objective is to characterize phytochrome and its functions in photosynthetically competent plants from molecular, biochemical and cellular perspectives. It is anticipated that others will continue to contribute indirectly to these efforts at the physiological level. The ultimate goal will be to develop this information from a comparative perspective in order to learn whether the different phytochromes have significantly different physicochemical properties, whether they fulfill independent functions and if so what these different functions are, and how each of the different phytochromes acts at primary molecular and cellular levels.

  12. Gene Expression Profiling in Peripheral Blood Cells and Synovial Membranes of Patients with Psoriatic Arthritis

    PubMed Central

    Barbieri, Alessandro; Patuzzo, Giuseppe; Tinazzi, Elisa; Argentino, Giuseppe; Beri, Ruggero; Lunardi, Claudio; Puccetti, Antonio

    2015-01-01

    Background Psoriatic arthritis (PsA) is an inflammatory arthritis whose pathogenesis is poorly understood; it is characterized by bone erosions and new bone formation. The diagnosis of PsA is mainly clinical and diagnostic biomarkers are not yet available. The aim of this work was to clarify some aspects of the disease pathogenesis and to identify specific gene signatures in paired peripheral blood cells (PBC) and synovial biopsies of patients with PsA. Moreover, we tried to identify biomarkers that can be used in clinical practice. Methods PBC and synovial biopsies of 10 patients with PsA were used to study gene expression using Affymetrix arrays. The expression values were validated by Q-PCR, FACS analysis and by the detection of soluble mediators. Results Synovial biopsies of patients showed a modulation of approximately 200 genes when compared to the biopsies of healthy donors. Among the differentially expressed genes we observed the upregulation of Th17 related genes and of type I interferon (IFN) inducible genes. FACS analysis confirmed the Th17 polarization. Moreover, the synovial trascriptome shows gene clusters (bone remodeling, angiogenesis and inflammation) involved in the pathogenesis of PsA. Interestingly 90 genes are modulated in both compartments (PBC and synovium) suggesting that signature pathways in PBC mirror those of the inflamed synovium. Finally the osteoactivin gene was upregulared in both PBC and synovial biopsies and this finding was confirmed by the detection of high levels of osteoactivin in PsA sera but not in other inflammatory arthritides. Conclusions We describe the first analysis of the trancriptome in paired synovial tissue and PBC of patients with PsA. This study strengthens the hypothesis that PsA is of autoimmune origin since the coactivity of IFN and Th17 pathways is typical of autoimmunity. Finally these findings have allowed the identification of a possible disease biomarker, osteoactivin, easily detectable in PsA serum. PMID

  13. Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis

    PubMed Central

    Wapelhorst, Britta; Grosser, Gary; Günther, Sabine; Alber, Jörg; Döring, Barbara; Kliesch, Sabine; Weidner, Wolfgang; Galuska, Christina E.; Hartmann, Michaela F.; Wudy, Stefan A.; Bergmann, Martin; Geyer, Joachim

    2013-01-01

    Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I–V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, β-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates. PMID:23667501

  14. Impaired Intestinal Calcium Absorption in Protein 4.1R-deficient Mice Due to Altered Expression of Plasma Membrane Calcium ATPase 1b (PMCA1b)*

    PubMed Central

    Liu, Congrong; Weng, Haibao; Chen, Lixiang; Yang, Shaomin; Wang, Hua; Debnath, Gargi; Guo, Xinhua; Wu, Liancheng; Mohandas, Narla; An, Xiuli

    2013-01-01

    Protein 4.1R was first identified in the erythrocyte membrane skeleton. It is now known that the protein is expressed in a variety of epithelial cell lines and in the epithelia of many tissues, including the small intestine. However, the physiological function of 4.1R in the epithelial cells of the small intestine has not so far been explored. Here, we show that 4.1R knock-out mice exhibited a significantly impaired small intestinal calcium absorption that resulted in secondary hyperparathyroidism as evidenced by increased serum 1,25-(OH)2-vitamin D3 and parathyroid hormone levels, decreased serum calcium levels, hyperplasia of the parathyroid, and demineralization of the bones. 4.1R is located on the basolateral membrane of enterocytes, where it co-localizes with PMCA1b (plasma membrane calcium ATPase 1b). Expression of PMCA1b in enterocytes was decreased in 4.1−/− mice. 4.1R directly associated with PMCA1b, and the association involved the membrane-binding domain of 4.1R and the second intracellular loop and C terminus of PMCA1b. Our findings have enabled us to define a functional role for 4.1R in small intestinal calcium absorption through regulation of membrane expression of PMCA1b. PMID:23460639

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    DOE PAGESBeta

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

    2016-01-21

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

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

    PubMed Central

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

    2016-01-01

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

  18. Elevated CTLA-4 expression on CD4 T cells from periodontitis patients stimulated with Porphyromonas gingivalis outer membrane antigen

    PubMed Central

    Aoyagi, T; Yamazaki, K; Kabasawa-Katoh, Y; Nakajima, T; Yamashita, N; Yoshie, H; Hara, K

    2000-01-01

    To characterize the T cell response to Porphyromonas gingivalis, we examined the expression of costimulatory molecules on T cells derived from adult periodontitis patients with high serum antibody titre to P. gingivalis. The expression of CD28, CTLA-4, CD40 ligand (CD40L) on CD4+ T cells was analysed by flow cytometry. IL-10 and transforming growth factor-beta (TGF-β) mRNA expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent image analysis. Peripheral blood mononuclear cells (PBMC) derived from periodontitis patients showed higher proliferative responses to P. gingivalis outer membrane (OM) than those from healthy controls (P < 0.05). The percentage of CTLA-4+ cells within CD4+ T cells of patients was significantly higher than that of healthy controls after P. gingivalis OM stimulation (33.0% versus 11.9%, P < 0.01). There was no significant difference in the percentages of CD28+ cells and CD40L+ cells, and the percentage of CD40L+ cells was low in both groups even after stimulation. Stimulation of PBMC with P. gingivalis OM induced significantly higher IL-10 mRNA expression in periodontitis patients than in healthy controls (P < 0.05). The level of TGF-β mRNA expression of patients tended to be higher than that of healthy controls, but there was no significant difference. To elucidate the functional role of CTLA-4, we further investigated the secondary proliferative response to P. gingivalis OM. Interestingly, P. gingivalis OM stimulation did not enhance antigen-specific secondary response. Anti-CTLA-4 MoAb had no effect on proliferation in the presence of P. gingivalis OM. CTLA-4Ig suppressed the proliferative response significantly (P < 0.01). These results suggest that T cell responses to P. gingivalis OM may be regulated by CTLA-4 that is expressed at the late phase of T cell activation, and, in part, immunosuppressive cytokines. Taken together, CTLA-4 may play a crucial role in the pathogenesis of chronic

  19. Developmental expression of calcium transport proteins in extraembryonic membranes of oviparous and viviparous Zootoca vivipara (Lacertilia, Lacertidae).

    PubMed

    Stewart, James R; Ecay, Tom W; Heulin, Benoit; Fregoso, Santiago P; Linville, Brent J

    2011-09-15

    The eggshell of oviparous lizards is a significant source of calcium for embryos, whereas the eggshell of viviparous lizards, when present, contains little calcium. In view of the potential cost to embryonic nutrition occasioned by the loss of eggshell calcium, the large number of independent origins of viviparity among lizards is surprising. Concomitant evolution of viviparity and calcium placentotrophy would ameliorate the loss of eggshell calcium, but a mechanism linking these events has yet to be discovered. Zootoca vivipara, a lizard with geographic variation in its mode of parity, is an excellent model for studying mechanisms of calcium transport to oviparous and viviparous embryos because each is highly dependent on calcium secreted by the uterus (eggshell or placenta) and ontogenetic patterns of embryonic calcium mobilization are similar. We compared developmental expression of the calcium transport protein calbindin-D(28K) in yolk splanchnopleure and chorioallantoic membranes of oviparous and viviparous embryos to test the hypothesis that the mechanism of calcium transport does not differ between modes of parity. We found that the ontogenetic pattern of protein expression is similar between reproductive modes and is correlated with calcium uptake from yolk and either eggshell or placenta. Calbindin-D(28K) is localized in the chorionic epithelium of embryos of both reproductive modes. These findings suggest that the embryonic calcium transport machinery is conserved in the transition between reproductive modes and that an adaptation of oviparous embryos for calcium uptake from eggshells functions similarly to transport calcium directly from uterine secretions. PMID:21865511

  20. Bismuth Dimercaptopropanol (BisBAL) Inhibits the Expression of Extracellular Polysaccharides and Proteins by Brevundimonas diminuta: Implications for Membrane Microfiltration

    SciTech Connect

    Badireddy, Appala R.; Chellam, Shankararaman; Yanina, Svetlana; Gassman, Paul L.; Rosso, Kevin M.

    2008-02-15

    A 2:1 molar ratio preparation of bismuth with a lipophilic dithiol (3-dimercapto-1-propanol, BAL)significantly reduced extracellular polymeric substances (EPS) expression by Brevundimonas diminuta in suspended cultures at levels just below the minimum inhibitory concentration (MIC). Total polysaccharides and proteins secreted by B. diminuta decreased by approximately 95% over a 5-day period when exposed to the bismuth-BAL chelate (BisBAL) at near MIC (12 μM). Fourier-transform infrared spectroscopy (FTIR) suggested that a possible mechanism of biofilm disruption by BisBAL is the inhibition of carbohydrate Oacetylation. FTIR also revealed extensive homology between EPS samples with and without BisBAL treatment, with proteins, polysaccharides, and peptides varying predominantly only in the amount expressed. EPS secretion decreased following BisBAL treatment as verified by atomic force microscopy and scanning electron microscopy. Without BisBAL treatment, a slime-like EPS matrix secreted by B. diminuta resulted in biofouling and inefficient hydrodynamic backwashing of microfiltration membranes.

  1. Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

    PubMed

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

  2. Ferlins Show Tissue-Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans-Golgi/Recycling Ferlins.

    PubMed

    Redpath, Gregory M I; Sophocleous, Reece A; Turnbull, Lynne; Whitchurch, Cynthia B; Cooper, Sandra T

    2016-03-01

    Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca(2+) -regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca(2+) -dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca(2+) -triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations. PMID:26707827

  3. Bismuth dimercaptopropanol (BisBAL) inhibits the expression of extracellular polysaccharides and proteins by Brevundimonas diminuta: implications for membrane microfiltration.

    PubMed

    Badireddy, Appala Raju; Chellam, Shankararaman; Yanina, Svetlana; Gassman, Paul; Rosso, Kevin M

    2008-02-15

    A 2:1 molar ratio preparation of bismuth with a lipophilic dithiol (3-dimercapto-1-propanol, BAL) significantly reduced extracellular polymeric substances (EPS) expression by Brevundimonas diminuta in suspended cultures at levels just below the minimum inhibitory concentration (MIC). Total polysaccharides and proteins secreted by B. diminuta decreased by approximately 95% over a 5-day period when exposed to the bismuth-BAL chelate (BisBAL) at near MIC (12 microM). Fourier-transform infrared spectroscopy (FTIR) suggested that a possible mechanism of biofilm disruption by BisBAL is the inhibition of carbohydrate O-acetylation. FTIR also revealed extensive homology between EPS samples with and without BisBAL treatment, with proteins, polysaccharides, and peptides varying predominantly only in the amount expressed. EPS secretion decreased following BisBAL treatment as verified by atomic force microscopy and scanning electron microscopy. Without BisBAL treatment, a slime-like EPS matrix secreted by B. diminuta resulted in biofouling and inefficient hydrodynamic backwashing of microfiltration membranes. PMID:17705249

  4. Construction and Immunogenicity of Recombinant Swinepox Virus Expressing Outer Membrane Protein L of Salmonella.

    PubMed

    Fang, Yizhen; Lin, Huixing; Ma, Zhe; Fan, Hongjie

    2016-07-28

    Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wildtype SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpLspecific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p < 0.05). The concentrations of IFN-γ and IL-4, which represent Th1-type and Th2-type cytokine responses, were significantly higher (p < 0.05) in the rSPVOmpL- vaccinated group than in the other three groups. After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpLvaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection. PMID:27012234

  5. Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii.

    PubMed

    Chi, Xiaoyuan; Zhang, Xiaowen; Guan, Xiangyu; Ding, Ling; Li, Youxun; Wang, Mingqing; Lin, Hanzhi; Qin, Song

    2008-04-01

    Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal delta 12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae). PMID:18545969

  6. The effects of phenotypic plasticity on photosynthetic performance in winter rye, winter wheat and Brassica napus.

    PubMed

    Dahal, Keshav; Kane, Khalil; Gadapati, Winona; Webb, Elizabeth; Savitch, Leonid V; Singh, Jasbir; Sharma, Pooja; Sarhan, Fathey; Longstaffe, Fred J; Grodzinski, Bernard; Hüner, Norman P A

    2012-02-01

    The contributions of phenotypic plasticity to photosynthetic performance in winter (cv Musketeer, cv Norstar) and spring (cv SR4A, cv Katepwa) rye (Secale cereale) and wheat (Triticum aestivum) cultivars grown at either 20°C [non-acclimated (NA)] or 5°C [cold acclimated (CA)] were assessed. The 22-40% increase in light-saturated rates of CO₂ assimilation in CA vs NA winter cereals were accounted for by phenotypic plasticity as indicated by the dwarf phenotype and increased specific leaf weight. However, phenotypic plasticity could not account for (1) the differential temperature sensitivity of CO₂ assimilation and photosynthetic electron transport, (2) the increased efficiency and light-saturated rates of photosynthetic electron transport or (3) the decreased light sensitivity of excitation pressure and non-photochemical quenching between NA and NA winter cultivars. Cold acclimation decreased photosynthetic performance of spring relative to winter cultivars. However, the differences in photosynthetic performances between CA winter and spring cultivars were dependent upon the basis on which photosynthetic performance was expressed. Overexpression of BNCBF17 in Brassica napus generally decreased the low temperature sensitivity (Q₁₀) of CO₂ assimilation and photosynthetic electron transport even though the latter had not been exposed to low temperature. Photosynthetic performance in wild type compared to the BNCBF17-overexpressing transgenic B. napus indicated that CBFs/DREBs regulate not only freezing tolerance but also govern plant architecture, leaf anatomy and photosynthetic performance. The apparent positive and negative effects of cold acclimation on photosynthetic performance are discussed in terms of the apparent costs and benefits of phenotypic plasticity, winter survival and reproductive fitness. PMID:21883254

  7. DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus.

    PubMed Central

    Ficht, T A; Bearden, S W; Sowa, B A; Adams, L G

    1989-01-01

    The cloning of the gene(s) encoding a 36-kilodalton (kDa) cell envelope protein of Brucella abortus has been previously described (T. A. Ficht, S. W. Bearden, B. A. Sowa, and L. G. Adams, Infect, Immun. 56:2036-2046, 1988). In an attempt to define the nature of the previously described duplication at this locus we have sequenced 3,500 base pairs of genomic DNA encompassing this region. The duplication represented two similar open reading frames which shared more than 85% homology at the nucleotide level but differed primarily because of the absence of 108 nucleotides from one of the two gene copies. These two genes were read from opposite strands and potentially encoded proteins which are 96% homologous. The predicted gene products were identical over the first 100 amino acids, including 22-amino-acid-long signal sequences. The amino acid composition of the predicted proteins was similar to that obtained for the Brucella porin isolated by Verstreate et al. (D. R. Verstreate, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982) and presumably represented two copies of the porin gene, tentatively identified as omp 2a (silent) and omp 2b (expressed). The homology between the two genes extended to and included Shine-Dalgarno sequences 7 base pairs upstream from the ATG start codons. Homology at the 3' ends extended only as far as the termination codon, but both genes had putative rho-independent transcription termination sites. Localization of the promoters proved more difficult, since the canonical procaryotic sequences could not be identified in the region upstream of either gene. Promoter activity was demonstrated by ligation to a promoterless lacZ gene in pMC1871. However, only one active promoter could be identified by using this system. A 36-kDa protein was synthesized in E. coli with the promoter in the native orientation and was identical in size to the protein produced in laboratory-grown B. abortus. When

  8. Characterization and expression of plasma and tonoplast membrane aquaporins in primed seed of Brassica napus during germination under stress conditions.

    PubMed

    Gao, Y P; Young, L; Bonham-Smith, P; Gusta, L V

    1999-07-01

    Two aquaporin genes were isolated from a cDNA library of canola (Brassica napus L.). The first aquaporin, BnPIP1 of 1094 bp, encoding a putative polypeptide of 287 amino acids with a predicted molecular mass of 30.4 kDa and a pI of 7.8, belongs to the family of plasma membrane intrinsic protein (PIPs) aquaporins. The B. napus aquaporin showed 85-94% identity to the Arabidopsis thaliana PIPs. ABA priming of seed induced high levels of BnPIP1 transcript which remained after subsequent re-drying of the seed. The second aquaporin, Bny-TIP2 of 1020 bp, encoded a putative polypeptide of 253 amino acids with a predicted molecular mass of 25.8 kDa and a pI of 5.8. Bngamma-TIP2 showed 83-90% identity to gamma-TIP genes from a variety of plant species. Bngamma-TIP2 was expressed only when radicle protrusion occurred in either untreated or primed seeds. Seeds primed with PEG or ABA germinated earlier and showed a higher final percentage of germination than unprimed seed, particularly under salt and osmotic stresses at low temperature. Transcripts of both BnPIP1 and Bngamma-TIP2 genes were present earlier during germination of primed seeds than non-primed seed. From these results, we conclude that BnPIP1 is related to the water transportation required for enzymatic metabolism of storage nutrients at the early stages of canola seed germination whereas Bngamma-TIP2 expression is related to cell growth associated with radicle protrusion. Priming induced the expression of BnPIP1 but had no effect on Bngamma-TIP2. PMID:10480387

  9. Quantifying reflectance anisotropy of photosynthetically active radiation in grasslands

    NASA Technical Reports Server (NTRS)

    Middleton, Elizabeth M.

    1992-01-01

    Quantifying the vegetative surface's reflectance anisotropy was an important part of the First ISLSCP Field Experiment, as its major objectives focused on retrieval of surface parameters from satellite-derived reflectances. The explicit remote measurements for approximating the bidirectional reflectance distribution function (BRDF) of photosynthetically active radiation had not been previously undertaken. In this paper the proper expression of reflectance for BRDFs for retrieval of canopy parameters is assessed.

  10. Dynamics of Membrane Potential Variation and Gene Expression Induced by Spodoptera littoralis, Myzus persicae, and Pseudomonas syringae in Arabidopsis

    PubMed Central

    Bricchi, Irene; Bertea, Cinzia M.; Occhipinti, Andrea; Paponov, Ivan A.; Maffei, Massimo E.

    2012-01-01

    Background Biotic stress induced by various herbivores and pathogens invokes plant responses involving different defense mechanisms. However, we do not know whether different biotic stresses share a common response or which signaling pathways are involved in responses to different biotic stresses. We investigated the common and specific responses of Arabidopsis thaliana to three biotic stress agents: Spodoptera littoralis, Myzus persicae, and the pathogen Pseudomonas syringae. Methodology/Principal Findings We used electrophysiology to determine the plasma membrane potential (Vm) and we performed a gene microarray transcriptome analysis on Arabidopsis upon either herbivory or bacterial infection. Vm depolarization was induced by insect attack; however, the response was much more rapid to S. littoralis (30 min −2 h) than to M. persicae (4–6 h). M. persicae differentially regulated almost 10-fold more genes than by S. littoralis with an opposite regulation. M. persicae modulated genes involved in flavonoid, fatty acid, hormone, drug transport and chitin metabolism. S. littoralis regulated responses to heat, transcription and ion transport. The latest Vm depolarization (16 h) was found for P. syringae. The pathogen regulated responses to salicylate, jasmonate and to microorganisms. Despite this late response, the number of genes differentially regulated by P. syringae was closer to those regulated by S. littoralis than by M. persicae. Conclusions/Significance Arabidopsis plasma membranes respond with a Vm depolarization at times depending on the nature of biotic attack which allow setting a time point for comparative genome-wide analysis. A clear relationship between Vm depolarization and gene expression was found. At Vm depolarization timing, M. persicae regulates a wider array of Arabidopsis genes with a clear and distinct regulation than S. littoralis. An almost completely opposite regulation was observed between the aphid and the pathogen, with the former

  11. Expression, purification and identification of an immunogenic fragment in the ectodomain of prostate-specific membrane antigen

    PubMed Central

    TAO, RONG; NI, ZHENHUA; LIU, CHONG; ZHU, MIN; JI, XIAOWEN; CHEN, XUEMIN; SHEN, JIANGFAN; TU, SHAOHUA

    2016-01-01

    The present study aimed to identify, express and purify an immunogenic fragment in the ectodomain of prostate-specific membrane antigen (PSMA) within a fusion protein. The PSMA amino acid sequence published in National Center for Biotechnology Information GenBank was used to determine sequence homology and immunogenic index analyses, additionally using BLASTN, Protean and ExPASy software to predict the polypeptide sequences of immunogenic epitopes. The gene sequence encoding the ectodomain of the polypeptide immunogenic fragments, containing the identified immunogenic epitopes, was generated using whole-gene synthesis. Prokaryotic expression vector pET-32a-r-ectodomain-PSMA was constructed and the recombinant plasmids were transformed into competent BL21 (DE3) Escherichia coli, which was followed by induction of recombinant protein expression using isopropyl-β-D-thiogalactopyranoside. Fusion proteins were isolated and purified using affinity chromatography and their immune activity was subsequently investigated using western blot analysis. Purified protein was used to immunize BALB/c mice in order to generate polyclonal antibodies, and the binding of polyclonal antibodies to prostate cancer cell lines in vitro was evaluated using flow cytometry. A total of 3 polypeptide fragments with high specificity were identified following analysis using numerous software packages, and the gene sequences encoding regions containing the 2 most immunogenic fragments were synthesized and successfully inserted into the prokaryotic expression vector pET-32a-r-ectodomain-PSMA. The recombinant PSMA protein fragment had a molecular weight of ~50 kDa and 95% purity. Western blot analysis revealed that the r-ectodomain-PSMA fusion protein specifically bound to the anti-PSMA ectodomain monoclonal antibody. Flow cytometry demonstrated that polyclonal antibodies raised against these recombinant proteins could specifically bind to PSMA-positive LNCaP cells, but not to PSMA-negative PC-3

  12. Triazine herbicide resistance in the photosynthetic bacterium Rhodopseudomonas sphaeroides

    SciTech Connect

    Brown, A.E.; Gilbert, C.W.; Guy, R.; Arntzen, C.J.

    1984-10-01

    The photoaffinity herbicide azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine) selectively labels the L subunit of the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides. Herbicide-resistant mutants retain the L subunit and have altered binding properties for methylthio- and chloro-substituted triazines as well as altered equilibrium constants for electron transfer between primary and secondary electron acceptors. We suggest that a subtle alteration in the L subunit is responsible for herbicide resistance and that the L subunit is the functional analog of the 32-kDa Q/sub B/ protein of chloroplast membranes. 42 references, 6 figures, 1 table.

  13. Photosynthetic redox imbalance governs leaf sectoring in the Arabidopsis thaliana variegation mutants immutans, spotty, var1, and var2.

    PubMed

    Rosso, Dominic; Bode, Rainer; Li, Wenze; Krol, Marianna; Saccon, Diego; Wang, Shelly; Schillaci, Lori A; Rodermel, Steven R; Maxwell, Denis P; Hüner, Norman P A

    2009-11-01

    We hypothesized that chloroplast energy imbalance sensed through alterations in the redox state of the photosynthetic electron transport chain, measured as excitation pressure, governs the extent of variegation in the immutans mutant of Arabidopsis thaliana. To test this hypothesis, we developed a nondestructive imaging technique and used it to quantify the extent of variegation in vivo as a function of growth temperature and irradiance. The extent of variegation was positively correlated (R(2) = 0.750) with an increase in excitation pressure irrespective of whether high light, low temperature, or continuous illumination was used to induce increased excitation pressure. Similar trends were observed with the variegated mutants spotty, var1, and var2. Measurements of greening of etiolated wild-type and immutans cotyledons indicated that the absence of IMMUTANS increased excitation pressure twofold during the first 6 to 12 h of greening, which led to impaired biogenesis of thylakoid membranes. In contrast with IMMUTANS, the expression of its mitochondrial analog, AOX1a, was transiently upregulated in the wild type but permanently upregulated in immutans, indicating that the effects of excitation pressure during greening were also detectable in mitochondria. We conclude that mutations involving components of the photosynthetic electron transport chain, such as those present in immutans, spotty, var1, and var2, predispose Arabidopsis chloroplasts to photooxidation under high excitation pressure, resulting in the variegated phenotype. PMID:19897671

  14. Photosynthetic Redox Imbalance Governs Leaf Sectoring in the Arabidopsis thaliana Variegation Mutants immutans, spotty, var1, and var2[W

    PubMed Central

    Rosso, Dominic; Bode, Rainer; Li, Wenze; Krol, Marianna; Saccon, Diego; Wang, Shelly; Schillaci, Lori A.; Rodermel, Steven R.; Maxwell, Denis P.; Hüner, Norman P.A.

    2009-01-01

    We hypothesized that chloroplast energy imbalance sensed through alterations in the redox state of the photosynthetic electron transport chain, measured as excitation pressure, governs the extent of variegation in the immutans mutant of Arabidopsis thaliana. To test this hypothesis, we developed a nondestructive imaging technique and used it to quantify the extent of variegation in vivo as a function of growth temperature and irradiance. The extent of variegation was positively correlated (R2 = 0.750) with an increase in excitation pressure irrespective of whether high light, low temperature, or continuous illumination was used to induce increased excitation pressure. Similar trends were observed with the variegated mutants spotty, var1, and var2. Measurements of greening of etiolated wild-type and immutans cotyledons indicated that the absence of IMMUTANS increased excitation pressure twofold during the first 6 to 12 h of greening, which led to impaired biogenesis of thylakoid membranes. In contrast with IMMUTANS, the expression of its mitochondrial analog, AOX1a, was transiently upregulated in the wild type but permanently upregulated in immutans, indicating that the effects of excitation pressure during greening were also detectable in mitochondria. We conclude that mutations involving components of the photosynthetic electron transport chain, such as those present in immutans, spotty, var1, and var2, predispose Arabidopsis chloroplasts to photooxidation under high excitation pressure, resulting in the variegated phenotype. PMID:19897671

  15. Carotenoids are essential for the assembly of cyanobacterial photosynthetic complexes.

    PubMed

    Tóth, Tünde N; Chukhutsina, Volha; Domonkos, Ildikó; Knoppová, Jana; Komenda, Josef; Kis, Mihály; Lénárt, Zsófia; Garab, Győző; Kovács, László; Gombos, Zoltán; van Amerongen, Herbert

    2015-10-01

    In photosynthetic organisms, carotenoids (carotenes and xanthophylls) are important for light harvesting, photoprotection and structural stability of a variety of pigment-protein complexes. Here, we investigated the consequences of altered carotenoid composition for the functional organization of photosynthetic complexes in wild-type and various mutant strains of the cyanobacterium Synechocystis sp. PCC 6803. Although it is generally accepted that xanthophylls do not play a role in cyanobacterial photosynthesis in low-light conditions, we have found that the absence of xanthophylls leads to reduced oligomerization of photosystems I and II. This is remarkable because these complexes do not bind xanthophylls. Oligomerization is even more disturbed in crtH mutant cells, which show limited carotenoid synthesis; in these cells also the phycobilisomes are distorted despite the fact that these extramembranous light-harvesting complexes do not contain carotenoids. The number of phycocyanin rods connected to the phycobilisome core is strongly reduced leading to high amounts of unattached phycocyanin units. In the absence of carotenoids the overall organization of the thylakoid membranes is disturbed: Photosystem II is not formed, photosystem I hardly oligomerizes and the assembly of phycobilisomes remains incomplete. These data underline the importance of carotenoids in the structural and functional organization of the cyanobacterial photosynthetic machinery. PMID:26045333

  16. Cetuximab-mediated cellular cytotoxicity is inhibited by HLA-E membrane expression in colon cancer cells.

    PubMed

    Levy, Estrella Mariel; Sycz, Gabriela; Arriaga, Juan Martin; Barrio, María Marcela; von Euw, Erika María; Morales, Sergio Bayo; González, Mariana; Mordoh, José; Bianchini, Michele

    2009-04-01

    Cetuximab, an anti-epidermal growth factor receptor monoclonal antibody, has been shown to increase the median survival of colorectal cancer patients. We previously reported that the expression of HLA-E is significantly increased in primary human colorectal cancer, perhaps contributing to tumour escape from immune surveillance. To establish if HLA-E could be a factor that renders colorectal cancer cells less susceptible to antibody-dependent cellular cytotoxicity (ADCC), in the present study we analysed Cetuximab-mediated cytotoxicity against several colorectal cancer cell lines expressing, or not, HLA-E at the cell surface. We first observed that colorectal cancer cells treated with Cetuximab were killed more efficiently by ADCC. Interestingly, treatment of target cells with recombinant human-beta2-microglobulin inhibits Cetuximab-mediated ADCC through HLA-E membrane stabilization. The specific immunosuppressive role of HLA-E was confirmed using an anti-NKG2A monoclonal antibody, that restored the ability of immune cells to kill their target. This result demonstrates that HLA-E at the cell surface can reliably suppress the ADCC effect. On the other hand, Cetuximab induced a direct growth inhibition but only at high concentrations; furthermore, the CDC effect was quite moderate, and we failed to observe a pro-apoptotic effect. Taking into account that our findings suggest that ADCC activity is the main anti-tumour effect observed at clinically achievable concentrations of Cetuximab at the tumour site, we suggest that determination of HLA-E in colorectal cancer could be relevant to predict success of Cetuximab treatment. PMID:19318419

  17. Function and expression study uncovered hepatocyte plasma membrane ecto-ATP synthase as a novel player in liver regeneration.

    PubMed

    Taurino, Federica; Giannoccaro, Caterina; Sardanelli, Anna Maria; Cavallo, Alessandro; De Luca, Elisa; Santacroce, Salvatore; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2016-08-15

    ATP synthase, canonically mitochondrially located, is reported to be ectopically expressed on the plasma membrane outer face of several cell types. We analysed, for the first time, the expression and catalytic activities of the ecto- and mitochondrial ATP synthase during liver regeneration. Liver regeneration was induced in rats by two-thirds partial hepatectomy. The protein level and the ATP synthase and/or hydrolase activities of the hepatocyte ecto- and mitochondrial ATP synthase were analysed on freshly isolated hepatocytes and mitochondria from control, sham-operated and partial hepatectomized rats. During the priming phase of liver regeneration, 3 h after partial hepatectomy, liver mitochondria showed a marked lowering of the ATP synthase protein level that was reflected in the impairment of both ATP synthesis and hydrolysis. The ecto-ATP synthase level, in 3 h partial hepatectomized hepatocytes, was decreased similarly to the level of the mitochondrial ATP synthase, associated with a lowering of the ecto-ATP hydrolase activity coupled to proton influx. Noteworthily, the ecto-ATP synthase activity coupled to proton efflux was completely inhibited in 3 h partial hepatectomized hepatocytes, even in the presence of a marked intracellular acidification that would sustain it as in control and sham-operated hepatocytes. At the end of the liver regeneration, 7 days after partial hepatectomy, the level and the catalytic activities of the ecto- and mitochondrial ATP synthase reached the control and sham-operated values. The specific modulation of hepatocyte ecto-ATP synthase catalytic activities during liver regeneration priming phase may modulate the extracellular ADP/ATP levels and/or proton influx/efflux trafficking, making hepatocyte ecto-ATP synthase a candidate for a novel player in the liver regeneration process. PMID:27287557

  18. Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

    PubMed Central

    Burdick, Monica M.; Reynolds, Nathan M.; Martin, Eric W.; Hawes, Jacquelyn V.; Carlson, Grady E.; Cuckler, Chaz M.; Bates, Michael C.; Barthel, Steven R.; Dimitroff, Charles J.

    2014-01-01

    Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization

  19. Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b.

    PubMed Central

    Munson, R; Tolan, R W

    1989-01-01

    The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2535836

  20. Potato tuber expression of Arabidopsis WRINKLED1 increase triacylglycerol and membrane lipids while affecting central carbohydrate metabolism.

    PubMed

    Hofvander, Per; Ischebeck, Till; Turesson, Helle; Kushwaha, Sandeep K; Feussner, Ivo; Carlsson, Anders S; Andersson, Mariette

    2016-09-01

    Tuber and root crops virtually exclusively accumulate storage products in the form of carbohydrates. An exception is yellow nutsedge (Cyperus esculentus) in which tubers have the capacity to store starch and triacylglycerols (TAG) in roughly equal amounts. This suggests that a tuber crop can efficiently handle accumulation of energy dense oil. From a nutritional as well as economic aspect, it would be of interest to utilize the high yield capacity of tuber or root crops for oil accumulation similar to yellow nutsedge. The transcription factor WRINKLED1 from Arabidopsis thaliana, which in seed embryos induce fatty acid synthesis, has been shown to be a major factor for oil accumulation. WRINKLED1 was expressed in potato (Solanum tuberosum) tubers to explore whether this factor could impact tuber metabolism. This study shows that a WRINKLED1 transcription factor could induce triacylglycerol accumulation in tubers of transformed potato plants grown in field (up to 12 nmol TAG/mg dry weight, 1% of dry weight) together with a large increase in polar membrane lipids. The changes in metabolism further affected starch accumulation and composition concomitant with massive increases in sugar content. PMID:26914183

  1. Expression of dopamine D2 receptor in PC-12 cells and regulation of membrane conductances by dopamine.

    PubMed

    Zhu, W H; Conforti, L; Millhorn, D E

    1997-10-01

    PC-12 cells depolarize during hypoxia and release dopamine. The hypoxia-induced depolarization is due to inhibition of an O2-sensitive K+ current. The role of dopamine released during hypoxia is uncertain, but it could act as an autocrine to modulate membrane conductance during hypoxia. The current study was undertaken to investigate this possibility. Reverse transcription-polymerase chain reaction and sequence analysis revealed that the D2 isoform of the dopamine receptor is expressed in rat PC-12 cells. Exogenously applied dopamine and the D2 agonist quinpirole elicited inhibition of a voltage-dependent K+ current (I(K)) that was prevented by sulpiride, a D2 receptor antagonist. Dopamine and quinpirole applied during hypoxia potentiated the inhibitory effect of hypoxia on I(K). We also found that quinpirole caused reversible inhibition of a voltage-dependent Ca2+ current (I(Ca)) and attenuation of the increase in intracellular free Ca2+ during hypoxia. Our results indicate that dopamine released from PC-12 cells during hypoxia acts via a D2 receptor to "autoregulate" I(K) and I(Ca). PMID:9357757

  2. Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites

    PubMed Central

    Yap, Alan; Azevedo, Mauro F; Gilson, Paul R; Weiss, Greta E; O’Neill, Matthew T; Wilson, Danny W; Crabb, Brendan S; Cowman, Alan F

    2014-01-01

    Summary Malaria is caused by obligate intracellular parasites, of which Plasmodium falciparum is the most lethal species. In humans, P. falciparum merozoites (invasive forms of the parasite) employ a host of parasite proteins to rapidly invade erythrocytes. One of these is the P. falciparum apical membrane antigen 1 (PfAMA1) which forms a complex with rhoptry neck proteins at the tight junction. Here, we have placed the Pfama1 gene under conditional control using dimerizable Cre recombinase (DiCre) in P. falciparum. DiCre-mediated excision of the loxP-flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle. This reduces growth by 40%, due to decreased invasion efficiency characterized by a post-invasion defect in sealing of the parasitophorous vacuole. These results show that PfAMA1 is an essential protein for merozoite invasion in P. falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event. PMID:24571085

  3. Two promoters and two translation start sites control the expression of the Shigella flexneri outer membrane protease IcsP

    PubMed Central

    Hensley, Christopher T.; Kamneva, Olga K.; Levy, Karen M.; Labahn, Stephanie K.; Africa, Lia A.; Wing, Helen J.

    2011-01-01

    The Shigella flexneri outer membrane protease IcsP proteolytically cleaves the actin-based motility protein IcsA from the bacterial surface. The icsP gene is monocistronic and lies downstream of an unusually large intergenic region on the Shigella virulence plasmid. In silico analysis of this region predicts a second transcription start site 84 bp upstream of the first. Primer extension analyses and beta-galactosidase assays demonstrate that both transcription start sites are used. Both promoters are regulated by the Shigella virulence gene regulator VirB and both respond similarly to conditions known to influence Shigella virulence gene expression (iron concentration, pH, osmotic pressure, and phase of growth). The newly identified promoter lies upstream of a Shine-Dalgarno sequence and second 5’-ATG-3’, which is in frame with the annotated icsP gene. The use of either translation start site leads to the production of IcsP capable of proteolytically cleaving IcsA. A bioinformatic scan of the Shigella genome reveals multiple occurrences of in-frame translation start sites associated with putative Shine –Dalgarno sequences, immediately upstream and downstream of annotated open reading frames. Taken together, our observations support the possibility that the use of in-frame translation start sites may generate different protein isoforms, thereby expanding the proteome encoded by bacterial genomes. PMID:21225241

  4. Expression of a Sensory Neuron Membrane Protein SNMP2 in Olfactory Sensilla of Codling Moth Cydia pomonella (Lepidoptera: Tortricidae).

    PubMed

    Huang, Xinglong; Liu, Lu; Fang, Yiqing; Feng, Jinian

    2016-08-01

    In insects, sensory neuron membrane proteins (SNMPs) are critical peripheral olfactory proteins and highly promote the sensitivity of pheromone detection. In this study, we cloned an SNMP transcript (CpomSNMP2, GenBank KU302714) from the antennae of the codling moth Cydia pomonella (L.) Its open reading frame is 1,575 bp and it encodes a protein with 524 amino acids. CpomSNMP2 contains two putative transmembrane domains and has a large extracellular loop. Phylogenetic analysis showed that CpomSNMP2 is clustered into the group of previously characterized lepidopteron SNMP2s. Expression levels of CpomSNMP2 were significantly higher in antennae of both males and females than in tissues from the thoraxes, abdomens, legs, and wings. CpomSNMP2 was distributed in sensilla trichodea of both males and females, but only in sensilla chaetica of males. This study provides evidence for olfactory roles of CpomSNMP2 in this moth. PMID:27329623

  5. Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

    PubMed Central

    Blanco, D R; Champion, C I; Exner, M M; Shang, E S; Skare, J T; Hancock, R E; Miller, J N; Lovett, M A

    1996-01-01

    We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure. PMID:8955283

  6. Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from Mycobacterium avium subsp. paratuberculosis Strains K-10 and 187▿†

    PubMed Central

    Radosevich, Thomas J.; Reinhardt, Timothy A.; Lippolis, John D.; Bannantine, John P.; Stabel, Judith R.

    2007-01-01

    Little is known of protein expression in Mycobacterium avium subsp. paratuberculosis and how this contributes to pathogenesis. In the present study, proteins from both membranes and cytosol were prepared from two strains of M. avium subsp. paratuberculosis, i.e., laboratory-adapted strain K-10 and a recent isolate, strain 187, obtained from a cow exhibiting clinical signs of Johne's disease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol and membrane proteins from K-10 and 187 showed marked differences in protein expression. Relative levels of protein expression from both M. avium subsp. paratuberculosis strains were measured by using amine-reactive isobaric tagging reagents (iTRAQ) and tandem mass spectroscopy. Protein identification and relative expression data were obtained for 874 membrane and cytosolic proteins from the M. avium subsp. paratuberculosis proteome. These data showed a number of significant differences in protein expression between strain K-10 and clinical isolate 187. Examples of proteins expressed at higher levels in clinical isolate 187 compared to strain K-10 are AtpC, RpoA, and several proteins involved in fatty acid biosynthesis. In contrast, proteins such as AhpC and several proteins involved in nitrogen metabolism were expressed at higher levels in strain K-10 compared to strain 187. These data may provide insights into the proteins whose expression is important in natural infection but are modified once M. avium subsp. paratuberculosis is adapted to laboratory cultivation. Results from these studies will provide tools for developing a better understanding of M. avium subsp. paratuberculosis infection in the host and offer potential as diagnostic reagents and vaccine candidates. PMID:17142399

  7. Targeted Expression of Stromelysin-1 in Mammary Gland Provides Evidence for a Role of Proteinases in Branching Morphogenesis and the Requirement for an Intact Basement Membrane for Tissue-specific Gene Expression

    SciTech Connect

    Sympson, Carolyn J; Talhouk, Rabih S; Alexander, Caroline M; Chin, Jennie R; Cliff, Shirley M; Bissell, Mina J; Werb, Zena

    1994-05-01

    The extracellular matrix (ECM) is an important regulator of the differentiated phenotype of mammary epithelial cells in culture. Despite the fact that ECM-degrading enzymes have been implicated in morphogenesis and tissue remodeling, there is little evidence for a direct role for such regulation in vivo. We generated transgenic mice that express autoactivated isoforms of the matrix metalloproteinase stromelysin-1, under the control of the whey acidic protein gene promoter, to examine the effect of inappropriate expression of this enzyme. Stromelysin-1 is implicated as the primary player in the loss of basement membrane and loss of function in the mammary gland during involution. The transgene was expressed at low levels in mammary glands of virgin female mice, leading to an unexpected phenotype: The primary ducts had supernumerary branches and showed precocious development of alveoli that expressed beta-casein at levels similar to that of an early- to mid-pregnant gland. Lactating glands showed high levels of transgene expression, with accumulation at the basement membrane, and a decrease in laminin and collagen IV, resulting in a loss of basement membrane integrity; this was accompanied by a dramatic alteration of alveolar morphology, with decreased size and shrunken lumina containing little beta-casein. During pregnancy, expression of endogenous whey acidic protein and beta-casein was reduced in transgenic glands, confirming the observed dependence of milk protein transcription of ECM in mammary epithelial cells in culture. These data provide direct evidence that stromelysin-1 activity can be morphogenic for mammary epithelial cells, inducing hyperproliferation and differentiation in virgin animals, and that its lytic activity can, indeed, disrupt membrane integrity and reduce mammary-specific function. We conclude that the balance of ECM-degrading enzymes with their inhibitors, and the associated regulation of ECM structure, is crucial for tissue-specific gene

  8. Polymorphisms in the Tumor Necrosis Factor Receptor Genes Affect the Expression Levels of Membrane-Bound Type I and Type II Receptors

    PubMed Central

    Sennikov, Sergey V.; Vasilyev, Filipp F.; Lopatnikova, Julia A.; Shkaruba, Nadezhda S.; Silkov, Alexander N.

    2014-01-01

    The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNFα receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNFα receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP −609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP −1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14+ monocytes compared to individuals with the GC genotype. The frequency differences in the CD3+ and CD19+ cells expressing TNFRII in relation to SNP −1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP −3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14+ cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI −609GT + TNFRII −3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNFα receptors and TNFα-mediated signaling. PMID:24782596

  9. Expression and insulin-regulated distribution of caveolin in skeletal muscle. Caveolin does not colocalize with GLUT4 in intracellular membranes.

    PubMed

    Muñoz, P; Mora, S; Sevilla, L; Kaliman, P; Tomàs, E; Gumà, A; Testar, X; Palacín, M; Zorzano, A

    1996-04-01

    Caveolin is believed to play an important role in sorting processes, vesicular trafficking, transmembrane signaling, and molecular transport across membranes. In this study we have evaluated the expression and distribution of caveolin in skeletal muscle and its interaction with GLUT4 glucose carriers. Caveolin was expressed to substantial levels in muscle and its expression was regulated in muscle; aging and high fat diet enhanced caveolin expression in skeletal muscle and inversely, myogenesis down-regulated caveolin in L6E9 cells. Under fasting conditions, most of caveolin was found in intracellular membranes and the caveolin present in the cell surface was found in both sarcolemma and T-tubules. Insulin administration led to a redistribution of caveolin from intracellular high density membrane fractions to intracellular lighter density fractions and to the cell surface; this pattern of insulin-induced redistribution was different to what was shown by GLUT4. These results suggests that caveolin is a component of an insulin-regulated machinery of vesicular transport in muscle. Quantitative immunoisolation of GLUT4 vesicles obtained from different intracellular GLUT4 populations revealed the absence of caveolin which substantiates the lack of colocalization of intracellular GLUT4 and caveolin. This indicates that caveolin is not involved in intracellular GLUT4 trafficking in skeletal muscle. PMID:8626501

  10. Structure, expression, and hormonal control of genes from the mosquito, Aedes aegypti, which encode proteins similar to the vitelline membrane proteins of Drosophila melanogaster.

    PubMed

    Lin, Y; Hamblin, M T; Edwards, M J; Barillas-Mury, C; Kanost, M R; Knipple, D C; Wolfner, M F; Hagedorn, H H

    1993-02-01

    Genomic and cDNA clones of a gene expressed after a blood meal in the mosquito, Aedes aegypti, were identified as having significant similarity to the vitelline membrane protein genes of Drosophila melanogaster. The predicted protein had unusually high contents of alanine, histidine, and proline and contained a region of hydrophobic amino acids that was highly conserved in the predicted protein of the D. melanogaster vitelline membrane protein genes. The 15a gene was expressed from 5 to 40 hr after a blood meal. It was expressed only in the follicle cells of the ovary, particularly in the cells surrounding the oocyte. The 15a gene was expressed in ovaries of the blood-fed, decapitated female in response to an injection of 20-hydroxyecdysone, and in ovaries from non-blood-fed females incubated with the hormone, even in the presence of cycloheximide. A second gene, with weaker homology to 15a, is presumably another member of a family of related genes, as is the case with D. melanogaster vitelline membrane protein genes. This second gene contained a coding sequence similar to a decapeptide recently isolated from mosquito ovaries as an "oostatic factor" (Borovsky et al., FASEB J. 4, 3015-3020, 1990). PMID:8432405

  11. Photosynthetic acclimation of the filamentous cyanobacterium, Plectonema boryanum UTEX 485, to temperature and light.

    PubMed

    Miśkiewicz, E; Ivanov, A G; Williams, J P; Khan, M U; Falk, S; Huner, N P

    2000-06-01

    Photosynthetic acclimation to temperature and irradiance was studied in the filamentous, non-heterocystous cyanobacterium Plectonema boryanum UTEX 485. Growth rates of this cyanobacterium measured at ambient CO2 were primarily influenced by temperature with minimal effects of irradiance. Both growth temperature and irradiance affected linolenic (18:3) and linoleic acid (18:2) levels in the four major lipid classes in an independent but additive manner. In contrast, photosynthetic acclimation was not due to either growth temperature or irradiance per se, but rather, due to the interaction of these environmental factors. P. boryanum grown at low temperature and moderate irradiance mimicked cells grown at high light. Compared to cells grown at either 29 degrees C/150 micromol m(-2) s(-1) (29/150) or 15/10, P. boryanum grown at either 15/150 or 29/750 exhibited: (1) reduced cellular levels of Chl a and phycobilisomes (PBS), and concomitantly higher content of an orange-red carotenoid, myxoxanthophyll; (2) higher light saturated rates (Pmax) when expressed on a Chl a basis but lower apparent quantum yields of oxygen evolution and (3) enhanced resistance to high light stress. P. boryanum grown at 15/150 regained normal blue-green pigmentation within 16 h after a temperature shift to 29 degrees C at a constant irradiance of 150 micromol m(-2) s(-1). DBMIB and KCN but not DCMU and atrazine partially inhibited the change in myxoxanthophyll/Chl a ratio following the shift from 15 to 29 degrees C. We conclude that P. boryanum responds to either varying growth temperature or varying growth irradiance by adjusting the ability to absorb light through decreasing the cellular contents of Chl a and light-harvesting pigments and screening of excessive light by myxoxanthophyll predominantly localized in the cell wall/cell membrane to protect PSII from over-excitation. The possible role of redox sensing/signalling for photosynthetic acclimation of cyanobacteria to either temperature

  12. Lack of Association between Membrane-Type 1 Matrix Metalloproteinase Expression and Clinically Relevant Molecular or Morphologic Tumor Characteristics at the Leading Edge of Invasive Colorectal Carcinoma.

    PubMed

    Arndt, Annette; Kraft, Klaus; Wardelmann, Eva; Steinestel, Konrad

    2015-01-01

    Colorectal cancer (CRC) is one of the leading causes of death from cancer in the western world, but tumor biology and clinical course show great interindividual variation. Molecular and morphologic tumor characteristics, such as KRAS/BRAF mutation status, mismatch repair (MMR) protein expression, tumor growth pattern, and tumor cell budding, have been shown to be of key therapeutic and/or prognostic relevance in CRC. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-anchored zinc-binding endopeptidase that is expressed at the leading edge of various invasive carcinomas and promotes tumor cell invasion through degradation of the extracellular matrix. The aim of this study was to investigate possible associations between MT1-MMP expression and molecular tumor characteristics as well as morphologic features of tumor aggressiveness in a consecutive series of 79 CRC tissue samples. However, although MT1-MMP was expressed in 41/79 samples (52%), there was no significant association between MT1-MMP expression and KRAS/BRAF mutation status, MMR protein expression, presence of lymphovascular invasion, tumor growth pattern, tumor-infiltrating lymphocytes, or tumor cell budding in our sample cohort (P > 0.05). Thus, we conclude that although MT1-MMP may play a role in CRC invasion, it is not of key relevance to the current models of CRC invasion and aggressiveness. PMID:26106602

  13. Membrane-tethered AKT kinase regulates basal synaptic transmission and early phase LTP expression by modulation of post-synaptic AMPA receptor level.

    PubMed

    Pen, Y; Borovok, N; Reichenstein, M; Sheinin, A; Michaelevski, I

    2016-09-01

    The serine/threonine kinase AKT/PKB plays a fundamental role in a wide variety of neuronal functions, including neuronal cell development, axonal growth, and synaptic plasticity. Multiple evidence link AKT signaling pathways to regulation of late phase long-term synaptic plasticity, synaptogenesis, and spinogenesis, as well as long-term memory formation. Nevertheless, the downstream effectors mediating the effects of AKT on early phase long-term potentiation (eLTP) are currently unknown. Here we report that using different regimes of pharmacological activation and inhibition of AKT activity in acute hippocampal slices, we found that AKT regulates the post-synaptic expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptors affecting solely the expression of eLTP, with no effect on its induction and maintenance. We further show that both maintenance of basal synaptic activity and expression of eLTP require plasma membrane tethering by activated AKT and that basal synaptic activity may be regulated via the direct effects of AKT1 on the expression level of post-synaptic AMPA receptors bypassing the canonical AKT signaling. Finally, we establish that eLTP expression requires the involvement of both the canonical AKT signaling pathways and the direct effect of AKT1 on AMPA receptor activity/expression in the post-synaptic membrane. © 2016 Wiley Periodicals, Inc. PMID:27068236

  14. Lack of Association between Membrane-Type 1 Matrix Metalloproteinase Expression and Clinically Relevant Molecular or Morphologic Tumor Characteristics at the Leading Edge of Invasive Colorectal Carcinoma

    PubMed Central

    Arndt, Annette; Kraft, Klaus; Wardelmann, Eva; Steinestel, Konrad

    2015-01-01

    Colorectal cancer (CRC) is one of the leading causes of death from cancer in the western world, but tumor biology and clinical course show great interindividual variation. Molecular and morphologic tumor characteristics, such as KRAS/BRAF mutation status, mismatch repair (MMR) protein expression, tumor growth pattern, and tumor cell budding, have been shown to be of key therapeutic and/or prognostic relevance in CRC. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-anchored zinc-binding endopeptidase that is expressed at the leading edge of various invasive carcinomas and promotes tumor cell invasion through degradation of the extracellular matrix. The aim of this study was to investigate possible associations between MT1-MMP expression and molecular tumor characteristics as well as morphologic features of tumor aggressiveness in a consecutive series of 79 CRC tissue samples. However, although MT1-MMP was expressed in 41/79 samples (52%), there was no significant association between MT1-MMP expression and KRAS/BRAF mutation status, MMR protein expression, presence of lymphovascular invasion, tumor growth pattern, tumor-infiltrating lymphocytes, or tumor cell budding in our sample cohort (P > 0.05). Thus, we conclude that although MT1-MMP may play a role in CRC invasion, it is not of key relevance to the current models of CRC invasion and aggressiveness. PMID:26106602

  15. Requirement of phosphatidylglycerol for maintenance of photosynthetic machinery.

    PubMed

    Sakurai, Isamu; Hagio, Miki; Gombos, Zoltan; Tyystjarvi, Taina; Paakkarinen, Virpi; Aro, Eva-Mari; Wada, Hajime

    2003-11-01

    Phosphatidylglycerol (PG) is a ubiquitous component of thylakoid membranes. Experiments with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 defective in biosynthesis of PG have demonstrated an indispensable role of PG in photosynthesis. In the present study, we have investigated the light susceptibility of the pgsA mutant with regard to the maintenance of the photosynthetic machinery. Growth of the mutant cells without PG increased the light susceptibility of the cells and resulted in severe photoinhibition of photosynthesis upon a high-light treatment, whereas the growth in the presence of PG was protected against photoinhibition. Photoinhibition induced by PG deprivation was mainly caused by an impairment of the restoration process. The primary target of the light-induced damage in thylakoid membranes, the D1 protein of photosystem (PS) II was, however, synthesized and degraded with similar rates irrespective of whether the mutant cells were incubated with PG or not. Intriguingly, it was found that instead of the synthesis of the D1 protein, the dimerization of the PSII core monomers was impaired in the PG-deprived mutant cells. Addition of PG to photoinhibited cells restored the dimerization capacity of PSII core monomers. These results suggest that PG plays an important role in the maintenance of the photosynthetic machinery through the dimerization and reactivation of the PSII core complex. PMID:14551333

  16. Cell membrane fatty acid changes and desaturase expression of Saccharomyces bayanus exposed to high pressure homogenization in relation to the supplementation of exogenous unsaturated fatty acids

    PubMed Central

    Serrazanetti, Diana I.; Patrignani, Francesca; Russo, Alessandra; Vannini, Lucia; Siroli, Lorenzo; Gardini, Fausto; Lanciotti, Rosalba

    2015-01-01

    Aims: The aim of this work was to study the responses of Saccharomyces bayanus cells exposed to sub-lethal high-pressure homogenization (HPH) and determine whether the plasmatic membrane can sense HPH in the presence, or absence, of exogenous unsaturated fatty acids (UFAs) in the growth medium. Methods and Results: High-pressure homogenization damaged and caused the collapse of cell walls and membranes of a portion of cells; however, HPH did not significantly affect S. bayanus cell viability (less than 0.3 Log CFU ml-1). HPH strongly affected the membrane fatty acid (FA) composition by increasing the percentage of total UFA when compared with saturated fatty acids. The gene expression showed that the transcription of OLE1, ERG3, and ERG11 increased after HPH. The presence of exogenous UFA abolished HPH-induced effects on the OLE1 and ERG3 genes, increased the percentage of membrane lipids and decreased the expression of OLE1 and ERG3 within 30 min of treatment. Conclusion: The results suggest a key role for UFA in the microbial cell response to sub-lethal stress. In addition, these data provide insight into the molecular basis of the response of S. bayanus to this innovative technology. Significance and Impact of the Study: Elucidation of the mechanism of action for sub-lethal HPH will enable the utilization of this technology to modulate the starter performance at the industrial scale. PMID:26528258

  17. Expression and adhesive ability of gicerin, a cell adhesion molecule, in the pock lesions of chorioallantoic membranes infected with an avian poxvirus.

    PubMed Central

    Tsukamoto, Y; Kotani, T; Hiroi, S; Egawa, M; Ogawa, K; Sasaki, F; Taira, E

    2001-01-01

    The expression and adhesive activities of gicerin, a cell adhesion protein, in the pock lesions on chicken chorioallantoic membranes (CAM) infected with an avian poxvirus were studied. In normal CAMs, gicerin was found on the flattened epithelial cells, and neurite outgrowth factor (NOF) was in the basement membrane. However, in the pock lesions on infected CAMs, gicerin was overexpressed on the cell membranes of hyperplastic epithelial cells forming thick epithelial layers. Neurite outgrowth factor was also found mainly in the basement membrane, but occasionally showed aberrant expression among hyperplastic cells. In vitro analyses, using the dissociated cells from pock lesions, demonstrated that an anti-gicerin polyclonal antibody inhibit cell aggregation activity and cell adhesion to NOF. These results suggest that gicerin might promote the cell-cell and cell-extracellular matrix protein bindings of the hyperplastic epithelial cells by its homophilic and heterophilic adhesive activities, and contribute to pock formation on the infected CAMs. Images Figure 1. Figure 2. Figure 3. Figure 5. PMID:11768132

  18. CD133 expression correlates with membrane beta-catenin and e-cadherin loss from human hair follicle placodes during morphogenesis

    PubMed Central

    Gay, Denise; Yang, Chao-Chun; Plikus, Maksim; Ito, Mayumi; Rivera, Charlotte; Treffeisen, Elsa; Doherty, Laura; Spata, Michelle; Millar, Sarah E.; Cotsarelis, George

    2014-01-01

    Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode ‘budding’ is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions is clearly required for budding. Snail-mediated downregulation of adherens junction component E-cadherin is important for placode budding in mice. Beta-catenin, another adherens junction component, has been more difficult to study due to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent adherens junction dissolution. PMID:25010141

  19. Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma MembraneV⃞

    PubMed Central

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-01-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth. PMID:16381811

  20. Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5.

    PubMed

    Rivera, M; Barillas-Mury, C; Christensen, K A; Little, J W; Wells, M A; Walker, F A

    1992-12-01

    The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver. PMID:1333795

  1. Reliable scale-up of membrane protein over-expression by bacterial auto-induction: from microwell plates to pilot scale fermentations.

    PubMed

    Deacon, Sarah E; Roach, Peter C J; Postis, Vincent L G; Wright, Gareth S A; Xia, Xiaobing; Phillips, Simon E V; Knox, J Paul; Henderson, Peter J F; McPherson, Michael J; Baldwin, Stephen A

    2008-12-01

    The production of well-ordered crystals of membrane proteins for structural investigation by X-ray diffraction typically requires extensive crystallization trials and may involve the screening of multiple detergents, lipids and other additives. Purification of sufficient amounts of protein for such trials is hampered by the fact that even when over-expressed, membrane proteins represent only a small percentage of the total protein content of bacteria. Fermentation-scale cultures of cells are therefore usually required. To maximize the efficiency and reduce the cost of such cultures, in the UK Membrane Protein Structure Initiative we have systematically investigated the use of auto-induction as an alternative to induction of expression with isopropyl-beta-D-thiogalactoside. We report here the benefits of first optimizing expression on a multiwell plate scale by systematically varying the concentrations of glucose, glycerol, lactose and succinate present in the auto-induction medium. For subsequent scale-up, comparison of isopropyl-beta-D-thiogalactoside induction in shake-flasks with auto-induction in shake-flasks and in 1L fermenters without and with control of pH and aeration revealed that highest yields of target protein were obtained using the latter culture conditions. However, analysis of the time-course of expression highlighted the importance of choosing the correct time for harvest. The high yields of target protein that can be obtained in a single batch by auto-induction, performed on a 30 l scale in a fermenter, obviate batch-to-batch variations that can add an unwanted variable to crystallization screening experiments. The approach described should therefore be of great utility for membrane protein production for structural studies. PMID:19023695

  2. Process for photosynthetically splitting water

    DOEpatents

    Greenbaum, Elias

    1984-01-01

    The invention is an improved process for producing gaseous hydrogen and oxygen from water. The process is conducted in a photolytic reactor which contains a water-suspension of a photoactive material containing a hydrogen-liberating catalyst. The reactor also includes a volume for receiving gaseous hydrogen and oxygen evolved from the liquid phase. To avoid oxygen-inactivation of the catalyst, the reactor is evacuated continuously by an external pump which circulates the evolved gases through means for selectively recovering hydrogen therefrom. The pump also cools the reactor by evaporating water from the liquid phase. Preferably, product recovery is effected by selectively diffusing the hydrogen through a heated semipermeable membrane, while maintaining across the membrane a magnetic field gradient which biases the oxygen away from the heated membrane. This promotes separation, minimizes the back-reaction of hydrogen and oxygen, and protects the membrane.

  3. The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation

    SciTech Connect

    Benning, C.; Somerville, C.R. ); Beatty, J.T. ); Prince, R.C. )

    1993-02-15

    All photosynthetic organisms, with the exception of several species of photosynthetic bacteria, are thought to contain the sulfolipid 6-sulfo-[alpha]-D-quinovosyldiacylglycerol. The association of this lipid with photosynthetic membranes has led to the assumption that it plays some role in photosynthesis. Stable null mutants of the photosynthetic bacterium Rhodobacter sphaeroides completely lacking sulfolipid were obtained by disruption of the sqdB gene. The ratios of the various components of the photosynthetic electron transport chain, as well as the electron transfer rates during cyclic electron transport, were not altered in the mutants, when grown under optimal conditions. Growth rates of wild type and mutants were identical under a variety of growth conditions, with the exception of phosphate limitation, which resulted in reduced growth of the mutants. Phosphate limitation of the wild type a used a significant reduction in the amount of all phospholipids and an increased amount of sulfolipid. By contrast, the sulfolipid-deficient mutant had reduced levels of phosphatidylcholine and phosphatidylethanolamine but maintained a normal level of phosphatidylglycerol. In addition, two unidentified lipids lacking phosphorus accumulated in the membranes of both wild-type and mutant strains under phosphate limitation. We conclude that sulfolipid plays no significant unique role in photoheterotrophic growth or photosynthetic electron transport in R. sphaeroides but may function as a surrogate for phospholipids, particularly phosphatidylglycerol, under phosphate-limiting conditions. 34 refs., 5 figs., 1 tab.

  4. Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation.

    PubMed

    Kabus, Armin; Georgi, Tobias; Wendisch, Volker F; Bott, Michael

    2007-05-01

    A critical factor in the biotechnological production of L: -lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP(+) via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by approximately 10% on glucose, approximately 70% on fructose, approximately 50% on glucose plus fructose, and even by approximately 300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type. PMID:17216441

  5. IMMUTANS Does Not Act as a Stress-Induced Safety Valve in the Protection of the Photosynthetic Apparatus of Arabidopsis during Steady-State Photosynthesis1

    PubMed Central

    Rosso, Dominic; Ivanov, Alexander G.; Fu, Aigen; Geisler-Lee, Jane; Hendrickson, Luke; Geisler, Matt; Stewart, Gregory; Krol, Marianna; Hurry, Vaughan; Rodermel, Steven R.; Maxwell, Denis P.; Hüner, Norman P.A.

    2006-01-01

    IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O2 to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P700 to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6×) and 16 (OE-16×) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25°C and photosynthetic photon flux density of 150 μmol photons m−2 s−1). Similar results were observed either after 3-d cold stress at 5°C or after full-leaf expansion at 5°C and photosynthetic photon flux density of 150 μmol photons m−2 s−1. Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25°C or 5°C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P700+ during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic

  6. IMMUTANS does not act as a stress-induced safety valve in the protection of the photosynthetic apparatus of Arabidopsis during steady-state photosynthesis.

    PubMed

    Rosso, Dominic; Ivanov, Alexander G; Fu, Aigen; Geisler-Lee, Jane; Hendrickson, Luke; Geisler, Matt; Stewart, Gregory; Krol, Marianna; Hurry, Vaughan; Rodermel, Steven R; Maxwell, Denis P; Hüner, Norman P A

    2006-10-01

    IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O(2) to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P(700) to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6 x) and 16 (OE-16 x) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1)). Similar results were observed either after 3-d cold stress at 5 degrees C or after full-leaf expansion at 5 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1). Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25 degrees C or 5 degrees C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P(700)(+) during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited

  7. Switch of Voltage-Gated K+ Channel Expression in the Plasma Membrane of Chondrogenic Cells Affects Cytosolic Ca2+-Oscillations and Cartilage Formation

    PubMed Central

    Matta, Csaba; Fodor, János; Katona, Éva; Bartok, Adam; Oláh, Tamás; Sebe, Attila; Csernoch, László; Panyi, Gyorgy; Zákány, Róza

    2011-01-01

    Background Understanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca2+-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation. Methodology/Principal Findings Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated NaV1.4, KV1.1, KV1.3 and KV4.1 channels, although KV1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of NaV1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K+ channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced KV currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca2+-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive KV1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive KV4.1 did not affect cartilage formation. Conclusions/Significance These data demonstrate that the differentiation and proliferation of chondrogenic cells depend

  8. Protection of gingival epithelium against complement-mediated damage by strong expression of the membrane attack complex inhibitor protectin (CD59).

    PubMed

    Rautemaa, R; Meri, S

    1996-01-01

    Adult periodontitis (AP) is a chronic inflammatory disease of the tooth-supporting apparatus. Activation products of the inflammation-inducing complement system have been detected in the gingival crevicular fluid at the site of gingival inflammation. In the present study, we examined whether evidence for ongoing complement activation in gingival tissues of patients with AP can be obtained. In light of the potential tissue-damaging effects of the complement system, we also examined how the gingival tissue is protected against the cytolytic activity of complement. Surgical and autopsy samples of AP (n = 18) and healthy (n = 11) gingiva were analyzed for the expression or deposition of the complement regulators protectin (CD59) and vitronectin (S-protein) and complement components C3d and C9 by indirect immunofluorescence microscopy with specific antibodies. In healthy gingiva, protection was strongly expressed on the membranes of epithelial cells and on the vascular endothelia of the underlying connective tissue. In AP, protectin was also strongly expressed by endothelial cells, but in the epithelia the expression was granular and weaker than in the healthy gingiva. Coarse granular deposits of complement components were seen in the subepithelial tissues of 61% (C3d), 39% (C9), and 33% (vitronectin) of AP patients, compared with 9% (one case in 11) in healthy controls. In addition, deposits of C3d, C9, and vitronectin were observed on the basement membranes of both pocket and oral epithelium of healthy and AP gingiva but not at sites of protectin expression. The results suggest an increased turnover of the complement system in the gingival tissues of AP patients. The gingival epithelium and connective tissue endothelia are well-protected against damage by the membrane attack complex of complement (MAC). Protection of the underlying connective tissue is insufficient, however, and may allow for deposition of MAC and autologous tissue damage in AP. PMID:8655761

  9. Salmonella enterica serotype Typhimurium ShdA is an outer membrane fibronectin-binding protein that is expressed in the intestine.

    PubMed

    Kingsley, Robert A; Santos, Renato L; Keestra, A Marijke; Adams, L Garry; Bäumler, Andreas J

    2002-02-01

    The shdA gene is the only determinant known to be required for persistence of Salmonella enterica serotype Typhimurium (S. Typhimurium) in the murine caecum and for efficient and prolonged shedding of the organism with the faeces. To study the biological activity of the ShdA protein, we examined its expression and binding activity. ShdA was not detected with anti-ShdA antiserum in S. Typhimurium strain ATCC14028 grown in vitro, suggesting that this protein is not expressed under standard conditions of bacterial cultivation in the laboratory. However, in mice infected with S. Typhimurium, an immunofluorescence signal detected with anti-ShdA antiserum co-localized with that generated by anti-O4 antiserum in thin sections from the caecum. Expression of the cloned shdA gene from the T7 promoter in vitro resulted in detection of ShdA in the outer membrane of S. Typhimurium and in binding of fibronectin to the bacterial surface. Binding of purified glutathione-S-transferase (GST)-ShdA fusion protein to fibronectin was dose dependent and could be partially inhibited by preincubation with antifibronectin antibodies. GST-ShdA bound to connective tissue and the basement membrane in thin sections from the murine caecum in situ. A similar labelling pattern was produced when thin sections of the murine caecum were stained with antifibronectin antiserum. Collectively, these data demonstrate that ShdA is a surface-localized, fibronectin-binding protein whose expression is induced in vivo in the murine caecum, a tissue in which a cognate receptor of this outer membrane protein is expressed. PMID:11929540

  10. Effects of Natural Flavonoids on Photosynthetic Activity and Cell Integrity in Microcystis aeruginosa

    PubMed Central

    Huang, Haomin; Xiao, Xi; Ghadouani, Anas; Wu, Jiaping; Nie, Zeyu; Peng, Cheng; Xu, Xinhua; Shi, Jiyan

    2015-01-01

    Flavonoids are natural polyphenolic compounds produced by many aquatic plants and released in their environments. In this study, the effects of several aquatic flavonoids on cyanobacterial Microcystis aeruginosa, especially in relation to the cell growth, photosynthetic activity, cell morphology, and cell membrane integrity, were investigated. Significant growth inhibition was observed when the cyanobacteria were exposed to three flavonoids, namely, 5,4'-dihydroxyflavone (DHF), apigenin, and luteolin. Luteolin reduced the effective quantum yield, photosynthetic efficiency, and maximal electron transport rate by 70%, 59% and 44%, respectively, whereas 5,4'-DHF and apigenin slightly affected these parameters, which implies that luteolin disrupts the photosynthetic system. Moreover, 5,4'-DHF and apigenin compromised the membrane integrity, and induced membrane depolarization in 52% and 38%, and permeabilization in 30% and 44% of the cells, respectively. The 5,4'-DHF and apigenin showed more pronounced effects on M. aeruginosa morphology and membrane integrity, compared to the luteolin. These results suggest that flavonoids could have significant effects on growth and physiological functions in cyanobacterial species. PMID:25584428

  11. Adaptation of the Photosynthetic Apparatus to Irradiance in Dunaliella tertiolecta1

    PubMed Central

    Sukenik, Assaf; Bennett, John; Mortain-Bertrand, Anne; Falkowski, Paul G.

    1990-01-01

    The time course of adaptation from a high to a low photon flux density was studied in the marine chlorophyte Dunaliella tertiolecta. A one-step transition from 700 to 70 micromole quanta per square meter per second resulted in a reduction of doubling rate from 1.1 to 0.4 per day within 24 hours, followed by a slower accumulation of photosynthetic pigments, light harvesting antenna complexes, Photosystem II reaction centers and structural lipids that constitute the thylakoid membranes. Photoregulated changes in the biochemical composition of the thylakoid proteins and lipids were functionally accompanied by decreases in the minimal photosynthetic quantum requirement and photosynthetic capacity, and an increase in the minimal turnover time for in vivo electron transport from water to CO2. Analysis of de novo synthesis of thylakoid membranes and proteins indicates that a high light to low light transition leads to a transient in carbon metabolism away from lipid biosynthesis toward the synthesis of the light harvesting antenna protein complexes, accompanied by a slower restoration rate of reaction centers and thylakoid membranes. This pattern of sequential synthesis of light harvesting complexes followed by reaction centers and membranes, appears to optimize light harvesting capabilities as cells adapt to low photon flux densities. Images Figure 2 Figure 7 Figure 8 PMID:16667402

  12. Photosynthetic responses to phytoplasma infection in Chinese jujube.

    PubMed

    Liu, Zhiguo; Zhao, Jin; Liu, Mengjun

    2016-08-01

    Phytoplasma is one of the most devastating plant pathogens. Jujube witches' broom (JWB) is a typical and highly fatal phytoplasma disease of Chinese jujube (Ziziphus jujuba Mill.), which is widely cultivated in Asia. To further elucidate the mechanism of plant-phytoplasma interaction, we first compared the effects of phytoplasma infection on photosynthetic pigments and activities between a JWB-resistant cultivar (Xingguang) and a susceptible cultivar (Pozao). Total chlorophyll and carotenoid levels were significantly decreased in the susceptible cultivar at later stages of infection, but were remarkably increased in the resistant cultivar at the earlier stages. Compared to uninfected plant, a significant decrease in the main photochemical parameters (Fv/Fm, ΦPSII and qP) was recorded at the initial stages of infection in the resistant cultivar, but occurred at later stages in the susceptible cultivar. Meanwhile, the qRT-PCR results of four key photosynthesis-related genes (ZjGluTR, ZjCBP, ZjRubisco and ZjRCA2) demonstrated that the expression patterns were similar in uninfected cultivars, but up-regulated in resistant cultivar and down-regulated in the susceptible one at 12 wks after grafting inoculation. Collectively, our data indicated that the resistant cultivar 'Xingguang' undergoes a decrease in initial stage (inhibiting phytoplasma multiplication) and then a rapid enhancement of photosynthetic activity (helping jujube recovery) in response to phytoplasma infection, while the susceptible cultivar 'Pozao' displays a later decrease in photosynthetic activity. The novel photosynthetic response pattern of the resistant cultivar may contribute to its stronger immunity to phytoplasma infection, which provides new insights into plant-phytoplasma interactions. PMID:27064193

  13. Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella.

    PubMed

    Fardini, Yann; Trotereau, Jérôme; Bottreau, Elisabeth; Souchard, Charlène; Velge, Philippe; Virlogeux-Payant, Isabelle

    2009-05-01

    In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the DeltabamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in DeltabamC and DeltadegP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coli and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As DeltasurA and DeltabamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD. PMID:19372159

  14. Hydrogen Biogeochemistry in Anaerobic and Photosynthetic Ecosystems

    NASA Technical Reports Server (NTRS)

    Hoehler, Tori M.; DeVincenzi, Donald L. (Technical Monitor)

    2000-01-01

    The simple biochemistry of molecular hydrogen is central to a large number of microbial processes, affecting the interaction of organisms with each other and with the environment. In anoxic sediments, a great majority of microbial redox processes involve hydrogen as a reactant, product or potential by-product. Accordingly, the energetics (thermodynamics) of each of these processes is affected by variations in local H2 concentrations. It has long been established that this effect is important in governing microbe-microbe interactions and there are multiple demonstrations that "interspecies hydrogen transfer" can alter the products of, inhibit/stimulate, or even reverse microbial metabolic reactions. In anoxic sediments, H2 concentrations themselves are thought to be controlled by the thermodynamics of the predominant H2-consuming microbial process. In sediments from Cape Lookout Bight, this relationship quantitatively describes the co-variation of H2 concentrations with temperature (for methanogens and sulfate reducers) and with sulfate concentration (for sulfate reducers). The quantitative aspect is import= for two reasons: 1) it permits the modeling of H2-sensitive biogeochemistry, such as anaerobic methane oxidation or pathways of organic matter remineralization, as a function of environmental controls; 2) for such a relationship to be observed requires that intracellular biochemistry and bioenergetics are being directly expressed in a component of the extracellular medium. H2 could therefore be utilized a non-invasive probe of cellular energetic function in intact microbial ecosystems. Based on the latter principle we have measured down-core profiles of H2 and other relevant physico-chemical parameters in order to calculate the metabolic energy yields (DG) that support microbial metabolism in Cape Lookout Bight sediments. Methanogens in this system apparently function with energy yields significantly smaller than the minimum requirements suggested by pure

  15. The OsLti6 genes encoding low-molecular-weight membrane proteins are differentially expressed in rice cultivars with contrasting sensitivity to low temperature.

    PubMed

    Morsy, Mustafa R; Almutairi, Abeer M; Gibbons, James; Yun, Song Joon; de Los Reyes, Benildo G

    2005-01-01

    Rice (Oryza sativa L.) is sensitive to chilling particularly at early stages of seedling establishment. Two closely related genes (OsLti6a, OsLti6b), which are induced by low temperature during seedling emergence were isolated from a cold tolerant temperate japonica rice cultivar. These genes are closely related to the Arabidopsis rare cold-inducible (RCI2) and barley low-temperature-inducible (BLT101) genes. Based on direct biochemical and indirect physiological evidence and similarity with a conserved protein domain in the Cluster of Orthologous Groups (COG) database (e.g., yeast PMP3), the rice genes belong to a class of low-molecular-weight hydrophobic proteins involved in maintaining the integrity of the plasma membrane during cold, dehydration and salt stress conditions. Both genes exhibit a genotype-specific expression signature characterized by early and late stress-inducible expression in tolerant and intolerant genotypes, respectively. The differences in temporal expression profiles are consistent with cultivar differences in cold-induced membrane leakiness and seedling vigor. The presence of CRT/DRE promoter cis-elements is consistent with the synchronized expression of OsLti6 genes with the C-repeat binding factor/drought responsive element-binding protein (CBF/DREB) transcriptional activator. The present results indicate that the Oslti6 genes are part of a battery of cold stress defense-related genes regulated by a common switch. PMID:15656983

  16. Photosynthetic light reactions--an adjustable hub in basic production and plant immunity signaling.

    PubMed

    Kangasjärvi, Saijaliisa; Tikkanen, Mikko; Durian, Guido; Aro, Eva-Mari

    2014-08-01

    Photosynthetic efficiency is a key trait that influences the sustainable utilization of plants for energy and nutrition. By now, extensive research on photosynthetic processes has underscored important structural and functional relationships among photosynthetic thylakoid membrane protein complexes, and their roles in determining the productivity and stress resistance of plants. Photosystem II photoinhibition-repair cycle, for example, has arisen vital in protecting also Photosystem I against light-induced damage. Availability of highly sophisticated genetic, biochemical and biophysical tools has greatly expanded the catalog of components that carry out photoprotective functions in plants. On thylakoid membranes, these components encompass a network of overlapping systems that allow delicate regulation of linear and cyclic electron transfer pathways, balancing of excitation energy distribution between the two photosystems and dissipation of excess light energy in the antenna system as heat. An increasing number of reports indicate that the above mentioned mechanisms also mediate important functions in the regulation of biotic stress responses in plants. Particularly the handling of excitation energy in the light harvesting II antenna complexes appears central to plant immunity signaling. Comprehensive understanding of the underlying mechanisms and regulatory cross-talk, however, still remain elusive. This review highlights the current understanding of components that regulate the function of photosynthetic light reactions and directly or indirectly also modulate disease resistance in higher plants. PMID:24361390

  17. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature.

    PubMed

    Liu, Wei; Li, Wei; He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  18. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature

    PubMed Central

    He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions. PMID:25894196

  19. The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases.

    PubMed

    Tsai, Chi-Neu; Tsai, Chia-Lung; Tse, Ka-Po; Chang, Hwan-You; Chang, Yu-Sun

    2002-07-23

    The latent membrane protein (LMP1) of Epstein-Barr virus (EBV) is expressed in EBV-associated nasopharyngeal carcinoma, which is notoriously metastatic. Although it is established that LMP1 represses E-cadherin expression and enhances the invasive ability of carcinoma cells, the mechanism underlying this repression remains to be elucidated. In this study, we demonstrate that LMP1 induces the expression and activity of the DNA methyltransferases 1, 3a, and 3b, using real-time reverse transcription-PCR and enzyme activity assay. This results in hypermethylation of the E-cadherin promoter and down-regulation of E-cadherin gene expression, as revealed by methylation-specific PCR, real-time reverse transcription-PCR and Western blotting data. The DNA methyltransferase inhibitor, 5'-Aza-2'dC, restores E-cadherin promoter activity and protein expression in LMP1-expressing cells, which in turn blocks cell migration ability, as demonstrated by the Transwell cell migration assay. Our findings suggest that LMP1 down-regulates E-cadherin gene expression and induces cell migration activity by using cellular DNA methylation machinery. PMID:12110730

  20. Enhanced Practical Photosynthetic CO2 Mitigation

    SciTech Connect

    Gregory Kremer; David J. Bayless; Morgan Vis; Michael Prudich; Keith Cooksey; Jeff Muhs

    2003-07-22

    This quarterly report documents significant achievements in the Enhanced Practical Photosynthetic CO{sub 2} Mitigation project during the period from 4/2/2003 through 7/01/2003. As indicated in the list of accomplishments below we have completed some long-term model scale bioreactor tests and are prepared to begin pilot scale bioreactor testing. Specific results and accomplishments for the second quarter of 2003 include: (1) Bioreactor support systems and test facilities: (a) Qualitative long-term survivability tests for S.C.1.2(2) on Omnisil have been successfully completed and results demonstrate a growth rate that appears to be acceptable. (b) Quantitative tests of long-term growth productivity for S.C.1.2(2) on Omnisil have been completed and initial results are promising. Initial results show that the mass of organisms doubled (from 54.9 grams to 109.8 grams) in about 5 weeks. Full results will be available as soon as all membranes and filters are completely dried. The growth rate should increase significantly with the initiation of weekly harvesting during the long term tests. (c) The phase 1 construction of the pilot scale bioreactor has been completed, including the solar collector and light distribution system. We are now in the phase of system improvement as we wait for CRF-2 results in order to be able to finalize the design and construction of the pilot scale system. (d) A mass transfer experimental setup was constructed in order to measure the mass transfer rate from the gas to the liquid film flowing over a membrane and to study the hydrodynamics of the liquid film flowing over a membrane in the bioreactor. Results were reported for mass transfer coefficient, film thickness, and fluid velocity over an Omnisil membrane with a ''drilled hole'' header pipe design. (2) Organisms and Growth Surfaces: (a) A selectivity approach was used to obtain a cyanobacterial culture with elevated resistance to acid pH. Microlonies of ''3.2.2 S.C.1 Positive'' migrated

  1. Kinetics of photosynthetic response to ultraviolet and photosynthetically active radiation in Synechococcus WH8102 (cyanobacteria).

    PubMed

    Fragoso, Glaucia M; Neale, Patrick J; Kana, Todd M; Pritchard, Alicia L

    2014-01-01

    The picoplanktonic cyanobacteria, Synechococcus spp., (Nägeli) are important contributors to global ocean primary production that can be stressed by solar radiation, both in the photosynthetically active (PAR) and ultraviolet (UV) range. We studied the responses of PSII quantum yield (active fluorescence), carbon fixation ((14)C assimilation) and oxygen evolution (membrane inlet mass spectrometry) in Synechococcus WH8102 under moderate UV and PAR. PSII quantum yield decreased during exposure to moderate UV and UV+PAR, with response to the latter being faster (6.4 versus 2.8 min, respectively). Repair processes were also faster when UV+PAR exposure was followed by moderate PAR (1.68 min response time) than when UV was followed by very low PAR (10.5 min response time). For the UV+PAR treatment, the initial decrease in quantum yield was followed by a 50% increase ("rebound") after 7 min exposure, showing an apparent photoprotection induction. While oxygen uptake increased with PAR, it did not change under UV, suggesting that this oxygen-dependent mechanism of photoprotection, which may be acting as an electron sink, is not an important strategy against UV. We used propyl gallate, an antioxidant, to test for plastid terminal oxidase (ptox) or ptox-like enzymes activity, but it caused nonspecific and toxic effects on Synechococcus WH8102. PMID:24175996

  2. PS2013 Satellite Workshop on Photosynthetic Light-Harvesting Systems

    SciTech Connect

    Niederman, Robert A.; Blankenship, Robert E.; Frank, Harry A.

    2015-02-07

    These funds were used for partial support of the PS2013 Satellite Workshop on Photosynthetic Light-Harvesting Systems, that was held on 8-11 August, 2013, at Washington University, St. Louis, MO. This conference, held in conjunction with the 16th International Congress on Photosynthesis/St. Louis, continued a long tradition of light-harvesting satellite conferences that have been held prior to the previous six international photosynthesis congresses. In this Workshop, the basis was explored for the current interest in replacing fossil fuels with energy sources derived form direct solar radiation, coupled with light-driven electron transport in natural photosynthetic systems and how they offer a valuable blueprint for conversion of sunlight to useful energy forms. This was accomplished through sessions on the initial light-harvesting events in the biological conversion of solar energy to chemically stored energy forms, and how these natural photosynthetic processes serve as a guide to the development of robust bio-hybrid and artificial systems for solar energy conversion into both electricity or chemical fuels. Organized similar to a Gordon Research Conference, a lively, informal and collegial setting was established, highlighting the exchange of exciting new data and unpublished results from ongoing studies. A significant amount of time was set aside for open discussion and interactive poster sessions, with a special session devoted to oral presentations by talented students and postdoctoral fellows judged to have the best posters. This area of research has seen exceptionally rapid progress in recent years, with the availability of a number of antenna protein structures at atomic resolution, elucidation of the molecular surface architecture of native photosynthetic membranes by atomic force microscopy and the maturing of ultrafast spectroscopic and molecular biological techniques for the investigation and manipulation of photosynthetic systems. The conferees

  3. Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.

    PubMed Central

    Connor, J; Bucana, C; Fidler, I J; Schroit, A J

    1989-01-01

    Phosphatidylserine (PS) is asymmetrically distributed in mammalian cell membranes, being preferentially localized in the inner leaflet. Some studies have suggested that a disturbance in the normal asymmetric distribution of PS--e.g., PS exposure in the outer leaflet of the cell membrane, which can occur upon platelet activation as well as in certain pathologic red cells--serves as a potent procoagulant surface and as a signal for triggering their recognition by macrophages. These studies suggest that the regulation of PS distribution in cell membranes may be critical in controlling coagulation and in determining the survival of pathologic cells in the circulation. In this paper we describe a sensitive technique, based on PS-dependent prothrombinase complex activity, for assessing the amount of PS on the external leaflet of intact viable cells. Our results indicate that tumorigenic, undifferentiated murine erythroleukemic cells express 7- to 8-fold more PS in their outer leaflet than do their differentiated, nontumorigenic counterparts. Increased expression of PS in the tumorigenic cells directly correlated with their ability to be recognized and bound by macrophages. PMID:2717615

  4. Regulation of Carotenoid Biosynthesis in Photosynthetic Organs.

    PubMed

    Llorente, Briardo

    2016-01-01

    A substantial proportion of the dazzling diversity of colors displayed by living organisms throughout the tree of life is determined by the presence of carotenoids, which most often provide distinctive yellow, orange and red hues. These metabolites play fundamental roles in nature that extend far beyond their importance as pigments. In photosynthetic lineages, carotenoids are essential to sustain life, since they have been exploited to maximize light harvesting and protect the photosynthetic machinery from photooxidative stress. Consequently, photosynthetic organisms have evolved several mechanisms that adjust the carotenoid metabolism to efficiently cope with constantly fluctuating light environments. This chapter will focus on the current knowledge concerning the regulation of the carotenoid biosynthetic pathway in leaves, which are the primary photosynthetic organs of most land plants. PMID:27485221

  5. Hybrid system of semiconductor and photosynthetic protein.

    PubMed

    Kim, Younghye; Shin, Seon Ae; Lee, Jaehun; Yang, Ki Dong; Nam, Ki Tae

    2014-08-29

    Photosynthetic protein has the potential to be a new attractive material for solar energy absorption and conversion. The development of semiconductor/photosynthetic protein hybrids is an example of recent progress toward efficient, clean and nanostructured photoelectric systems. In the review, two biohybrid systems interacting through different communicating methods are addressed: (1) a photosynthetic protein immobilized semiconductor electrode operating via electron transfer and (2) a hybrid of semiconductor quantum dots and photosynthetic protein operating via energy transfer. The proper selection of materials and functional and structural modification of the components and optimal conjugation between them are the main issues discussed in the review. In conclusion, we propose the direction of future biohybrid systems for solar energy conversion systems, optical biosensors and photoelectric devices. PMID:25091409

  6. The 4p16.3 Parkinson Disease Risk Locus Is Associated with GAK Expression and Genes Involved with the Synaptic Vesicle Membrane

    PubMed Central

    Nagle, Michael W.; Latourelle, Jeanne C.; Labadorf, Adam; Dumitriu, Alexandra; Hadzi, Tiffany C.; Beach, Thomas G.; Myers, Richard H.

    2016-01-01

    Genome-wide association studies (GWAS) have identified the GAK/DGKQ/IDUA region on 4p16.3 among the top three risk loci for Parkinson’s disease (PD), but the specific gene and risk mechanism are unclear. Here, we report transcripts containing the 3’ clathrin-binding domain of GAK identified by RNA deep-sequencing in post-mortem human brain tissue as having increased expression in PD. Furthermore, carriers of 4p16.3 PD GWAS risk SNPs show decreased expression of one of these transcripts, GAK25 (Gencode Transcript 009), which correlates with the expression of genes functioning in the synaptic vesicle membrane. Together, these findings provide strong evidence for GAK clathrin-binding- and J-domain transcripts’ influence on PD pathogenicity, and for a role for GAK in regulating synaptic function in PD. PMID:27508417

  7. Molecular characterization of rice OsBIANK1, encoding a plasma membrane-anchored ankyrin repeat protein, and its inducible expression in defense responses.

    PubMed

    Zhang, Xinchun; Li, Dayong; Zhang, Huijuan; Wang, Xiaoe; Zheng, Zhong; Song, Fengming

    2010-02-01

    A rice gene, OsBIANK1, encoding a protein containing a typical ankyrin repeat domain, was cloned and identified. The OsBIANK1 protein, consisting of 329 amino acids, contains a conserved ankyrin repeat domain with two ankyrin repeats organized in tandem and was showed to be localized on cytoplasmic membrane during transient expression in onion epidermal cells. Expression of OsBIANK1 was induced by treatment with benzothiadiazole (BTH), a chemical inducer capable of inducing disease resistance response in rice. In BTH-treated rice seedlings, expression of OsBIANK1 was further induced by infection with Magnaporthe grisea, the rice blast fungus, as compared with those in water-treated seedlings. Our preliminary results confirm previous evidences that OsBIANK1 may be involved in regulation of disease resistance response in rice. PMID:19288292

  8. Prioritization of copper for the use in photosynthetic electron transport in developing leaves of hybrid poplar

    PubMed Central

    Shahbaz, Muhammad; Ravet, Karl; Peers, Graham; Pilon, Marinus

    2015-01-01

    Plastocyanin (PC) is an essential and abundant copper (Cu) protein required for photosynthesis in higher plants. Severe copper deprivation has the potential to cause a defect in photosynthetic electron transport due to a lack in PC. The Cu-microRNAs, which are up-regulated under Cu deficiency, down-regulate the expression of target Cu proteins other than PC, cytochrome-c oxidase and the ethylene receptors. It has been proposed that this mechanism saves Cu for PC maturation. We aimed to test how hybrid poplar, a species that has capacity to rapidly expand its photosynthetically active tissue, responds to variations in Cu availability over time. Measurement of chlorophyll fluorescence after Cu depletion revealed a drastic effect on photosynthesis in hybrid poplar. The decrease in photosynthetic capacity was correlated with a reduction in PC protein levels. Compared to older leaves, PC decreased more strongly in developing leaves, which also lost more photosynthetic electron transport capacity. The effect of Cu depletion on older and more developed leaves was minor and these leaves maintained much of their photosynthetic capacity. Interestingly, upon resupply of Cu to the medium a very rapid recovery of Cu levels was seen in the younger leaves with a concomitant rise in the expression and activity of PC. In contrast, the expression of those Cu proteins, which are targets of microRNAs was under the same circumstances delayed. At the same time, Cu resupply had only minor effects on the older leaves. The data suggest a model where rapid recovery of photosynthetic capacity in younger leaves is made possible by a preferred allocation of Cu to PC in younger leaves, which is supported by Cu-microRNA expression. PMID:26089828

  9. Parameters of photosynthetic energy partitioning.

    PubMed

    Lazár, Dušan

    2015-03-01

    Almost every laboratory dealing with plant physiology, photosynthesis research, remote sensing, and plant phenotyping possesses a fluorometer to measure a kind of chlorophyll (Chl) fluorescence induction (FLI). When the slow Chl FLI is measured with addition of saturating pulses and far-red illumination, the so-called quenching analysis followed by the so-called relaxation analysis in darkness can be realized. These measurements then serve for evaluation of the so-called energy partitioning, that is, calculation of quantum yields of photochemical and of different types of non-photochemical processes. Several theories have been suggested for photosynthetic energy partitioning. The current work aims to summarize all the existing theories, namely their equations for the quantum yields, their meaning and their assumptions. In the framework of these theories it is also found here that the well-known NPQ parameter ( [Formula: see text] ; Bilger and Björkman, 1990) equals the ratio of the quantum yield of regulatory light-induced non-photochemical quenching to the quantum yield of constitutive non-regulatory non-photochemical quenching (ΦNPQ/Φf,D). A similar relationship is also found here for the PQ parameter (ΦP/Φf,D). PMID:25569797

  10. Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins.

    PubMed Central

    Dragic, T; Picard, L; Alizon, M

    1995-01-01

    Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry. PMID:7815477

  11. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation

    PubMed Central

    Lillemeier, Björn F; Mörtelmaier, Manuel A; Forstner, Martin B; Huppa, Johannes B; Groves, Jay T; Davis, Mark M

    2010-01-01

    The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dual-color fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane–associated signal transduction. PMID:20010844

  12. A stable, reusable, and highly active photosynthetic bioreactor by bio-interfacing an individual cyanobacterium with a mesoporous bilayer nanoshell.

    PubMed

    Jiang, Nan; Yang, Xiao-Yu; Deng, Zhao; Wang, Li; Hu, Zhi-Yi; Tian, Ge; Ying, Guo-Liang; Shen, Ling; Zhang, Ming-Xi; Su, Bao-Lian

    2015-05-01

    An individual cyanobacterium cell is interfaced with a nanoporous biohybrid layer within a mesoporous silica layer. The bio-interface acts as an egg membrane for cell protection and growth of outer shell. The resulting bilayer shell provides efficient functions to create a single cell photosynthetic bioreactor with high stability, reusability, and activity. PMID:25641812

  13. Purple non-sulfur photosynthetic bacteria monitor environmental stresses.

    PubMed

    Kis, Mariann; Sipka, Gábor; Asztalos, Emese; Rázga, Zsolt; Maróti, Péter

    2015-10-01

    Heavy metal ion pollution and oxygen deficiency are major environmental risks for microorganisms in aqueous habitat. The potential of purple non-sulfur photosynthetic bacteria for biomonitoring and bioremediation was assessed by investigating the photosynthetic capacity in heavy metal contaminated environments. Cultures of bacterial strains Rhodobacter sphaeroides, Rhodospirillum rubrum and Rubrivivax gelatinosus were treated with heavy metal ions in micromolar (Hg(2+)), submillimolar (Cr(6+)) and millimolar (Pb(2+)) concentration ranges. Functional assays (flash-induced absorption changes and bacteriochlorophyll fluorescence induction) and electron micrographs were taken to specify the harmful effects of pollution and to correlate to morphological changes of the membrane. The bacterial strains and functional tests showed differentiated responses to environmental stresses, revealing that diverse mechanisms of tolerance and/or resistance are involved. The microorganisms were vulnerable to the prompt effect of Pb(2+), showed weak tolerance to Hg(2+) and proved to be tolerant to Cr(6+). The reaction center controlled electron transfer in Rvx. gelatinosus demonstrated the highest degree of resistance against heavy metal exposure. PMID:26232748

  14. SANS Investigation of the Photosynthetic Machinery of Chloroflexus aurantiacus

    PubMed Central

    Tang, Kuo-Hsiang; Urban, Volker S.; Wen, Jianzhong; Xin, Yueyong; Blankenship, Robert E.

    2010-01-01

    Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements. PMID:20959079

  15. SANS Investigation of the Photosynthetic Machinery of Chloroflexus Aurantiacus

    SciTech Connect

    Tang, Kuo-Hsiang; Urban, Volker S; Jianzhong, Wen; Yueyong, Xin; Blankenship, Robert E

    2010-01-01

    Green photosynthetic bacteria harvest light and perform photosynthesis in low light environments, and contain specialized antenna complexes to adapt to this condition. In this report, we present studies using small-angle neutron scattering (SANS) to elucidate structural information about the photosynthetic apparatus, including the peripheral light harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contract variation in SANS measurments, our studies suggest that the B808-866 comples is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation for the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size for the isolated B808-866 complex is also suggested via dynamic light scattering measurements. Alos, a smaller size of the RC of Cfx. aurantiacus that the RC of the purple bacteria is observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.

  16. Photosynthetic Apparatus Formation during the Cell Cycle of Chlorella

    PubMed Central

    Venediktov, Pavel S.; Chemeris, Yuree K.; Grishina, Natalia A.

    1981-01-01

    Synchronous cell division in cultures of Chlorella vulgaris Beijerinck was induced by intermittent illumination: 9 hours light, 6 hours darkness. The rate of photosynthetic O2 evolution per cell increases 4-fold in a one-step manner at the beginning of the light period, to the same extent as the increase in cell number. Over the division cycle, the following accumulation times during the light period were found: chlorophyll a, between 2 and 8 hours, chlorophyll b, between 5 and 8 hours, reaction centers of photosystems I and II, between 2 and 6 hours; and cytochrome f, between 2.5 and 5 hours. Cytochrome f accumulation is closely followed by an increase in amplitude of the rapid phase in light-induced absorption increase at 520 nanometers and in intensity of the delayed light emission. Enhancement of the delayed fluorescence yield per flash under continuous illumination (caused by the establishment of the pH difference across the thylakoid membrane) is maximal by the first hour of the light period. These findings, and others described in the text, suggested that the 4-fold growth of photosynthetic apparatus in the course of the cell cycle cannot be the result of gradual rise of electron-transport chain number. Rather, it is the result of a series of successive syntheses of its individual components. The rate-limiting step of electron transport is probably located between plastoquinone and cytochrome f. PMID:16661795

  17. Psychological stress impairs Na+-dependent glucose absorption and increases GLUT2 expression in the rat jejunal brush-border membrane.

    PubMed

    Boudry, Gaëlle; Cheeseman, Christopher I; Perdue, Mary H

    2007-02-01

    Chronic psychological stress impacts many functions of the gastrointestinal tract. However, the effect of stress on nutrient absorption is poorly documented. This study was designed to investigate glucose transporters in rats submitted to different periods of water-avoidance stress (WAS). Rats were subjected to WAS (1 h/day) for 1, 5, or 10 consecutive days. Four hours after the last WAS session, rats were killed and segments of jejunum were mounted in Ussing chambers to study electrophysiological properties of the jejunum and Na+-dependent glucose absorption kinetics. Mucosa was obtained to prepare brush-border membrane vesicles (BBMV) used to measure [14C]fructose uptake as well as sodium-glucose transporter 1 (SGLT-1) and GLUT2 expression by Western blot analysis. Exposure of animals to WAS induced a decrease in Na+-dependent glucose absorption Vmax after 1, 5, and 10 days without any change in SGLT-1 expression. Potential difference across the jejunum was decreased for all stressed groups. Furthermore, we observed an increase in phloretin-sensitive uptake of [14C]fructose by BBMV after 1, 5, or 10 days of WAS, which was not present in control animals. This suggested the abnormal appearance of GLUT2 in the brush border, which was confirmed by Western blot analysis. We concluded that psychological stress induces major changes in glucose transport with a decrease in Na+-dependent glucose absorption and an increase in GLUT2 expression at the brush-border membrane level. PMID:17053095

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

    PubMed

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

    2006-12-29

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

  19. ELK3 Expression Correlates With Cell Migration, Invasion, and Membrane Type 1-Matrix Metalloproteinase Expression in MDA-MB-231 Breast Cancer Cells.

    PubMed

    Heo, Sun-Hee; Lee, Je-Yong; Yang, Kyung-Min; Park, Kyung-Soon

    2015-01-01

    ELK3 is a member of the Ets family of transcription factors. Its expression is associated with angiogenesis, vasculogenesis, and chondrogenesis. ELK3 inhibits endothelial migration and tube formation through the regulation of MT1-MMP transcription. This study assessed the function of ELK3 in breast cancer (BC) cells by comparing its expression between basal and luminal cells in silico and in vitro. In silico analysis showed that ELK3 expression was higher in the more aggressive basal BC cells than in luminal BC cells. Similarly, in vitro analysis showed that ELK3 mRNA and protein expression was higher in basal BC cells than in normal cells and luminal BC cells. To investigate whether ELK3 regulates basal cell migration or invasion, knockdown was achieved by siRNA in the basal BC cell line MDA-MB-231. Inhibition of ELK3 expression decreased cell migration and invasion and downregulated MT1-MMP, the expression of which is positively correlated with tumor cell invasion. In silico analysis revealed that ELK3 expression was associated with that of MT1-MMP in several BC cell lines (0.98 Pearson correlation coefficient). Though MT1-MMP expression was upregulated upon ELK3 nuclear translocation, ELK3 did not directly bind to the 1.3-kb promoter region of the MT1-MMP gene. These results suggest that ELK3 plays a positive role in the metastasis of BC cells by indirectly regulating MT1-MMP expression. PMID:26637400

  20. Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer membrane protein OmpL32

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer membrane proteins has been shown to modulate the effectiveness of the host immu...

  1. Ectopic expression of plasma membrane targeted subunits of the Ndc80-complex as a tool to study kinetochore biochemistry.

    PubMed

    Holmström, Tim H; Rehnberg, Jonathan; Ahonen, Leena J; Kallio, Marko J

    2009-06-01

    Genomic stability depends on the normal function of the kinetochore, a multi-protein assemblage, which consists of over 80 molecules including both constitutive and transiently binding components. Information regarding the spatial-temporal assembly of kinetochore subcomplexes is often limited by technical difficulties in their isolation. To study kinetochore subcomplex formation, we targeted separately Hec1 and Spc24, two subunits of the Ndc80 kinetochore compilation, to the plasma membrane by fusing them with the amino-terminal palmitoylation and myristoylation (pm) sequence of the receptor tyrosine kinase Fyn. We found that in early mitotic cells, pm-GFP-Hec1 and pm-GFP-Spc24 fusion proteins localised to the plasma membrane and were able to recruit all subunits of the Ndc80 complex (Ndc80/Hec1, Nuf2, Spc24 and Spc25) to these foci. In interphase cells, only Hec1-Nuf2 and Spc24-Spc25 heterodimers accumulated to the plasma membrane foci. The results propose that the assembly of Ndc80 tetramer can take place outside of the kinetochore but requires co-factors that are only present in mitotic cells. These findings provide the first experimental evidence on the successful employment of the plasma membrane targeting technique in the study of kinetochore biochemistry. PMID:19393581

  2. Effect of egg weight on composition, embryonic growth, and expression of amino acid transporter genes in yolk sac membranes and small intestines of the domestic pigeon (Columba livia).

    PubMed

    Chen, M X; Li, X G; Yan, H C; Wang, X Q; Gao, C Q

    2016-06-01

    The objective of this study was to investigate the effect of egg weight on the composition of the egg, the growth of the embryo, and the expression of amino acid transporter genes in the yolk sac membranes and small intestines of the domestic pigeon (Columba livia). A total of 240 fertilized eggs were collected and divided into two groups based on the weight of the eggs, light (LE) and heavy (HE). The composition of 20 eggs from each group was measured, and the remaining eggs were weighed and placed in an incubator. On embryonic days (E) 9, 11, 13, and 15 and day of hatch (DOH), 15 embryos/hatchlings from each group were measured for embryonic growth, and samples were collected. The HE had heavier yolk and albumen weights than the LE (P < 0.01). Compared with the LE, the HE had heavier yolk-free embryonic body and yolk sac weights from E13 to DOH (P < 0.05). Additionally, the HE had larger yolk sac membrane weights from E13 to E15 (P < 0.05) and had more residual yolk sac content on DOH than those of the LE (P < 0.01). The yolk absorption was greater for the HE than for the LE from E11 to E13 (P < 0.05). Furthermore, the abundance of CAT2 and PepT1 mRNA in the yolk sac membranes was greater in the HE than in the LE on E13 (P < 0.05). Compared with the LE, the gene expression of EAAT2 in the intestine on E13 was greater in the HE, whereas the expression of EAAT3 was lower in the HE (P < 0.05). Taken together, our results suggest that egg weight influenced the composition of the eggs, embryonic development, and expression of amino acid transporter genes in the yolk sac membranes and small intestines of pigeon embryos. PMID:26957627

  3. Structure, Function and Reconstitution of Antenna Complexes of Green Photosynthetic Bacteria

    SciTech Connect

    Blankenship, Robert E.

    2005-06-10

    Most chlorophyll-type pigments in a photosynthetic organism function as an antenna, absorbing light and transferring excitations to a photochemical reaction center where energy storage takes place by a series of chemical reactions. The green photosynthetic bacteria are characterized by large antenna complexes known as chlorosomes, in which pigment-pigment interactions are of dominant importance. The overall objective of this project is to determine the mechanisms of excitation transfer and regulation of this unique antenna system, including how it is integrated into the rest of the photosynthetic energy transduction apparatus. Techniques that are being used in this research include biochemical analysis, spectroscopy, microscopy, X-ray structural studies, and reconstitution from purified components. Our recent results indicate that the chlorosome baseplate structure, which is the membrane attachment site for the chlorosome to the membrane, is a unique pigment-protein that contains large amounts of carotenoids and small amounts of bacteriochlorophyll a. Reconstitution of directed energy transfer in chlorosomes will be carried out using purified baseplates and oligomeric pigments. The integral membrane B808-866 antenna complex from Chloroflexus aurantiacus and the Fenna-Matthews-Olson protein-reaction center complex from green sulfur bacteria will be characterized by spectroscopic and structural techniques.

  4. Effects of Cu deficiency on photosynthetic electron transport

    SciTech Connect

    Droppa, M.; Terry, N.; Horvath, G.

    1984-04-01

    The role of copper (Cu) in photosynthetic electron transport was explored by using Cu deficiency in sugar beet as an experimental approach. Copper influenced electron transport at two sites in addition to plastocyanin. Under mild deficiency (0.84 nmol of Cu per cm/sup 2/ of leaf area), electron transport between the two photosystems (PS) is inhibited but not electron transport within PS I or PS II measured separately. The chlorophyll/plastoquinone ratio was normal in Cu-deficient plants. However, the breakpoint in the Arrhenius plot of electron transport was shifted towards a higher temperature. It is concluded that Cu is necessary to maintain the appropriate membrane fluidity to ensure the mobility of plastoquinone molecules to transfer electrons between the two photosystems. Under severe deficiency (0.22 nmol of Cu per cm/sup 2/ of leaf area) both PS II and PS I electron transports were inhibited and to the same extent. PS II electron transport activity could not be restored by adding artifical electron donors. Polypeptides with M/sub r/s of 28,000 and 13,500 were missing in Cu-deficient chloroplast membranes. In PS II particles prepared from normal chloroplasts of spinach, 2 atoms of Cu per reaction center are present. We conclude that Cu influences PS II electron transport either directly, by participation in electron transfer as a constituent of an electron carrier, or indirectly, via the polypeptide composition of the membrane in the PS II complex.

  5. Modulation of MicroRNA Cluster miR-183-96-182 Expression by Epstein-Barr Virus Latent Membrane Protein 1

    PubMed Central

    Oussaief, Lassad; Fendri, Ali; Chane-Woon-Ming, Béatrice; Poirey, Remy; Delecluse, Henri-Jacques; Joab, Irène

    2015-01-01

    ABSTRACT Epstein-Barr virus (EBV) is an oncogenic human herpesvirus involved in the pathogenesis of Burkitt's lymphoma (BL) and various other lymphoproliferative disorders. In BL, EBV protein expression is restricted to EBV nuclear antigen 1 (EBNA1), but small noncoding RNAs such as EBV-encoded small RNAs (EBERs) and microRNAs (miRNAs) can also be detected. miRNAs play major roles in crucial processes such as proliferation, differentiation, and cell death. It has recently become clear that alterations in the expression profile of miRNAs contribute to the pathogenesis of a number of malignancies. During latent infection, EBV expresses 25 viral pre-miRNAs and modulates the expression of specific cellular miRNAs, such as miR-155 and miR-146, which potentially play a role in oncogenesis. Here, we established the small-RNA expression profiles of three BL cell lines. Using large-scale sequencing coupled to Northern blotting and real-time reverse transcription-PCR (RT-PCR) analysis validation, we demonstrated the differential expression of some cellular and viral miRNAs. High-level expression of the miR-183-96-182 cluster and EBV miR-BamHI A rightward transcript (miR-BART) cluster was significantly associated with EBV type I latency. This expression was not affected by viral reactivation since transforming growth factor β1 (TGF-β1) stimulation did not significantly change the miRNA profiles. However, using several approaches, including de novo infection with a mutant virus, we present evidence that the expression of latent membrane protein 1 (LMP-1) triggered downregulation of the expression of the miR-183-96-182 cluster. We further show that this effect involves the Akt signaling pathway. IMPORTANCE In addition to expressing their own miRNAs, herpesviruses also impact the expression levels of cellular miRNAs. This regulation can be either positive or negative and usually results in the perturbation of pathways to create a cellular environment that is more

  6. Damage and protection of the photosynthetic apparatus from UV-B radiation. II. Effect of quercetin at different pH.

    PubMed

    Dobrikova, Anelia G; Apostolova, Emilia L

    2015-07-20

    The effect of the exogenously added quercetin against the UV-B inhibition of the photosystem II (PSII) functions in isolated pea thylakoid membranes suspended at different pH of the medium (6.5, 7.6 and 8.4) was investigated. The data revealed that the interaction of this flavonoid with the membranes depends on the pH and influences the initial S0-S1 state distribution of PSII in the dark, the energy transfer between pigment-protein complexes of the photosynthetic apparatus and the membrane fluidity. Quercetin also displays a different UV-protective effect depending on its location in the membranes, as the effect is more pronounced at pH 8.4 when it is located at the membrane surface. The results suggest that quercetin induces structural changes in thylakoid membranes, one of the possible reasons for its protection of the photosynthetic apparatus. PMID:26282614

  7. Structural organization of photosynthetic apparatus in agranal chloroplasts of maize.

    PubMed

    Romanowska, Elzbieta; Kargul, Joanna; Powikrowska, Marta; Finazzi, Giovanni; Nield, Jon; Drozak, Anna; Pokorska, Berenika

    2008-09-19

    We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment. PMID:18632664

  8. Analysis of transcriptional regulation and tissue-specific expression of Avicennia marina Plasma Membrane Protein 3 suggests it contributes to Na(+) transport and homoeostasis in A. marina.

    PubMed

    Chidambaram, Rajalakshmi; Venkataraman, Gayatri; Parida, Ajay

    2015-07-01

    Plasma membrane proteins (PMP3) play a role in cation homoeostasis. The 5' flanking sequence of stress inducible, Avicennia marina PMP3 (AmPMP3prom) was transcriptionally fused to (a) GUS or (b) GFP-AmPMP3 and analyzed in transgenic tobacco. Tissue-histochemical GUS and GFP:AmPMP3 localization are co-incident under basal and stress conditions. AmPMP3prom directed GUS activity is highest in roots. Basal transcription is conferred by a 388bp segment upstream of the translation start site. A 463bp distal enhancer in the AmPMP3prom confers enhanced expression under salinity in all tissues and also responds to increases in salinity. The effect of a central, stem-specific negative regulatory region is suppressed by the distal enhancer. The A. marina rhizosphere encounters dynamic changes in salinity at the inter-tidal interface. The complex, tissue-specific transcriptional responsiveness of AmPMP3 to salinity appears to have evolved in response to these changes. Under salinity, guard cell and phloem-specific expression of GFP:AmPMP3 is highly enhanced. Mesophyll, trichomes, bundle sheath, parenchymatous cortex and xylem parenchyma also show GFP:AmPMP3 expression. Cis-elements conferring stress, root and vascular-specific expression are enriched in the AmPMP3 promoter. Pronounced vascular-specific AmPMP3 expression suggests a role in salinity induced Na(+) transport, storage, and secretion in A. marina. PMID:26025523

  9. Purification of the extracellular domain of the membrane protein GlialCAM expressed in HEK and CHO cells and comparison of the glycosylation.

    PubMed

    Gaudry, Jean-Philippe; Arod, Christian; Sauvage, Christophe; Busso, Stephane; Dupraz, Philippe; Pankiewicz, Renata; Antonsson, Bruno

    2008-03-01

    Adhesion molecules are essential for a wide range of biological and physiological functions, including cell-cell interactions, cell interactions with the extracellular matrix, cell migration, proliferation and survival. Defects in cell adhesion have been associated with pathological conditions such as neoplasia, and neurodegenerative diseases. We have identified a new adhesion molecule of the immunoglobulin family, GlialCAM. The same protein was recently published under the name hepaCAM and was suggested to be associated with hepatocellular carcinoma. Here we have expressed and purified the extracellular domain of this molecule in two mammalian expression systems, HEK and CHO cells. A three step purification protocol gave an over 95% pure protein. The extracellular domain of GlialCAM possesses several potential N- and O-glycosylation sites. Glycosylation is one of the most common post-translational modifications of secreted proteins and of the extracellular domains of membrane bound proteins. It can influence both the activity and the stability of the protein. The glycosylation pattern has been shown to depend on the cell type where the protein is expressed. We examined if differences in the glycosylation of this protein could be detected when it was expressed in the two commonly used mammalian expression systems, HEK and CHO. Differences in the glycosylation were detected. PMID:18082421

  10. Hyaluronic acid promotes the expression of progesterone receptor membrane component 1 via epigenetic silencing of miR-139-5p in human and rat granulosa cells.

    PubMed

    Zhao, Guangfeng; Zhou, Xue; Fang, Ting; Hou, Yayi; Hu, Yali

    2014-11-01

    Primary ovarian insufficiency (POI) is a serious reproductive dysfunction in which the follicle pool is reduced and depleted. Abnormal apoptosis of ovarian granulosa cells (GCs) is believed to result in follicle loss. Progesterone receptor membrane component 1 (PGRMC1), which is critical for GC survival, was reported to be reduced in POI patients, but the mechanism is unknown. In the present study, we found that PGRMC1 expression was correlated with the level of hyaluronic acid (HA) in POI patients. HA up-regulated PGRMC1 expression in GCs via suppression of miR-139-5p, which was proven by Western blotting and luciferase reporter assays to target PGRMC1. Consistent with these findings, levels of miR-139-5p were significantly increased and presented an inverse correlation with PGRMC1 in POI patients. Noticeably, HA inhibited CD44-mediated miR-139-5p expression but had no effect on luciferase activity after insertion of miR-139 promoter into luciferase plasmid. Interestingly, miR-139-5p was significantly up-regulated in KGN cells (GC tumor cell line) by the histone deacetylase inhibitor trichostatin A, indicating that HA down-regulated miR-139-5p expression via histone deacetylation. Taken together, we report an unrecognized mechanism of HA in the promotion of PGRMC1 expression, suggesting that HA may be a potential molecule for the prevention and treatment of POI. PMID:25232020

  11. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation

    SciTech Connect

    Kauss, M. Ariel; Reiterer, Gudrun; Bunaciu, Rodica P.; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G{sub 1} to S to G{sub 2}/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G{sub 0} cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation.

  12. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation.

    PubMed

    Kauss, M Ariel; Reiterer, Gudrun; Bunaciu, Rodica P; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G(1) to S to G(2)/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G(0) cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation. PMID:18692045

  13. Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines

    PubMed Central

    Kitabatake, T.; Moaddel, R.; Cole, R.; Gandhari, M.; Frazier, C.; Hartenstein, J.; Rosenberg, A.; Bernier, M.; Wainer, I. W.

    2008-01-01

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [3H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric α7 nicotinic acetylcholine receptors (α7 nAChR) and heteromeric nicotinic acetylcholine receptors (αxβy nAChRs), which was confirmed by the addition of subtype-specific inhibitors, κ-bungarotoxin (α7 nAChR) and K-bungarotoxin (αxβy nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), γ-aminobutyric acid (GABAA) and N-methyl-d-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABAA receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  14. Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines.

    PubMed

    Kitabatake, T; Moaddel, R; Cole, R; Gandhari, M; Frazier, C; Hartenstein, J; Rosenberg, A; Bernier, M; Wainer, I W

    2008-11-15

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  15. Up-converted fluorescence from photosynthetic light-harvesting complexes linearly dependent on excitation intensity.

    PubMed

    Leiger, Kristjan; Freiberg, Arvi

    2016-01-01

    Weak up-converted fluorescence related to bacteriochlorophyll a was recorded from various detergent-isolated and membrane-embedded light-harvesting pigment-protein complexes as well as from the functional membranes of photosynthetic purple bacteria under continuous-wave infrared laser excitation at 1064 nm, far outside the optically allowed singlet absorption bands of the chromophore. The fluorescence increases linearly with the excitation power, distinguishing it from the previously observed two-photon excited fluorescence upon femtosecond pulse excitation. Possible mechanisms of this excitation are discussed. PMID:25764015

  16. Regulation of Photosynthetic Electron Transport and Photoinhibition

    PubMed Central

    Roach, Thomas; Krieger-Liszkay, Anja Krieger

    2014-01-01

    Photosynthetic organisms and isolated photosystems are of interest for technical applications. In nature, photosynthetic electron transport has to work efficiently in contrasting environments such as shade and full sunlight at noon. Photosynthetic electron transport is regulated on many levels, starting with the energy transfer processes in antenna and ending with how reducing power is ultimately partitioned. This review starts by explaining how light energy can be dissipated or distributed by the various mechanisms of non-photochemical quenching, including thermal dissipation and state transitions, and how these processes influence photoinhibition of photosystem II (PSII). Furthermore, we will highlight the importance of the various alternative electron transport pathways, including the use of oxygen as the terminal electron acceptor and cyclic flow around photosystem I (PSI), the latter which seem particularly relevant to preventing photoinhibition of photosystem I. The control of excitation pressure in combination with the partitioning of reducing power influences the light-dependent formation of reactive oxygen species in PSII and in PSI, which may be a very important consideration to any artificial photosynthetic system or technical device using photosynthetic organisms. PMID:24678670

  17. A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli.

    PubMed

    Kovacic, Filip; Bleffert, Florian; Caliskan, Muttalip; Wilhelm, Susanne; Granzin, Joachim; Batra-Safferling, Renu; Jaeger, Karl-Erich

    2016-05-01

    Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of β-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137-His258-Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield. PMID:27419054

  18. Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice

    PubMed Central

    Yamamoto-Mizuma, Shintaro; Wang, Ge-Xin; Liu, Luis L; Schegg, Kathleen; Hatton, William J; Duan, Dayue; Horowitz, Burton; Lamb, Fred S; Hume, Joseph R

    2004-01-01

    ClC-3, a member of the large superfamily of ClC voltage-dependent Cl– channels, has been proposed as a molecular candidate responsible for volume-sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC-3 disrupted (Clcn3−/−) mice casts considerable doubt on this proposed role for ClC-3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3+/+ and Clcn3−/− mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3−/− mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock-out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3+/+ and Clcn3−/− mice are remarkably similar in terms of activation and inactivation kinetics, steady-state current densities, rectification, anion selectivity (I− > Cl− ≫ Asp−) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3−/− mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti-ClC-3 antibodies, and differential sensitivities to [ATP]i and free [Mg2+]i. These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3−/− mice

  19. Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

    PubMed

    Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

    1992-12-01

    Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197. PMID:1447140

  20. Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

    PubMed Central

    Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

    1992-01-01

    Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197. Images PMID:1447140

  1. Transgenic rice expressing a cassava (Manihot esculenta Crantz) plasma membrane gene MePMP3-2 exhibits enhanced tolerance to salt and drought stresses.

    PubMed

    Yu, Y; Cui, Y C; Ren, C; Rocha, P S C F; Peng, M; Xu, G Y; Wang, M L; Xia, X J

    2016-01-01

    Plasma membrane proteolipid 3 (PMP3) is a class of small hydrophobic proteins found in many organisms including higher plants. Some plant PMP3 genes have been shown to respond to abiotic stresses and to participate in the processes of plant stress tolerance. In this study, we isolated the cassava (Manihot esculenta Crantz) MePMP3-2 gene and functionally characterized its role in tolerance to abiotic stress by expressing it in rice (Oryza sativa L.). MePMP3-2 encodes a 77-amino acid protein belonging to a subgroup of plant PMP3s that have long hydrophylic C-terminal tails of unknown function. In silico analysis and co-localization studies indicated that MePMP3-2 is a plasma membrane protein with two transmembrane domains, similar to other PMP3s. In cassava leaves, MePMP3-2 expression was up-regulated by salt and drought stresses. Heterologous constitutive expression of MePMP3-2 in rice did not alter plant growth and development but increased tolerance to salt and drought stresses. In addition, under stress conditions MePMP3-2 transgenic plants accumulated less malondialdehyde, had increased levels of proline, and exhibited greater up-regulation of the stress-related genes OsProT and OsP5CS, but led to only minor changes in OsDREB2A and OsLEA3 expression. These findings indicate that MePMP3-2 may play an important role in salt and drought stress tolerance in transgenic rice. PMID:26909954

  2. The tolC locus of Escherichia coli affects the expression of three major outer membrane proteins.

    PubMed Central

    Morona, R; Reeves, P

    1982-01-01

    tolC mutants, which are resistant to colicin E1 and also highly sensitive to detergents and dyes, were shown to lack the OmpF outer membrane protein. There was little effect on transcription as judged by the use of an ompF-lac operon fusion strain, and the tolC effect was probably due to a post-transcriptional effect. The NmpC protein and protein 2 were also tolC dependent. Images PMID:6281230

  3. Engineering of cyanobacteria for the photosynthetic production of limonene from CO2.

    PubMed

    Kiyota, Hiroshi; Okuda, Yukiko; Ito, Michiho; Hirai, Masami Yokota; Ikeuchi, Masahiko

    2014-09-20

    Isoprenoids, major secondary metabolites in many organisms, are utilized in various applications. We constructed a model photosynthetic production system for limonene, a volatile isoprenoid, using a unicellular cyanobacterium that expresses the plant limonene synthase. This system produces limonene photosynthetically at a nearly constant rate and that can be efficiently recovered using a gas-stripping method. This production does not affect the growth of the cyanobacteria and is markedly enhanced by overexpression of three enzymes in the intrinsic pathway to provide the precursor of limonene, geranyl pyrophosphate. The photosynthetic production of limonene in our system is more or less sustained from the linear to stationary phase of cyanobacterial growth for up to 1 month. PMID:24905149

  4. Regulation of gene expression through a transcriptional repressor that senses acyl-chain length in membrane phospholipids.

    PubMed

    Hofbauer, Harald F; Schopf, Florian H; Schleifer, Hannes; Knittelfelder, Oskar L; Pieber, Bartholomäus; Rechberger, Gerald N; Wolinski, Heimo; Gaspar, Maria L; Kappe, C Oliver; Stadlmann, Johannes; Mechtler, Karl; Zenz, Alexandra; Lohner, Karl; Tehlivets, Oksana; Henry, Susan A; Kohlwein, Sepp D

    2014-06-23

    Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription. PMID:24960695

  5. Effect of latent membrane protein 1 expression on overall survival in Epstein-Barr virus-associated cancers: a literature-based meta-analysis

    PubMed Central

    Tang, Ling-Long; Mao, Yan-Ping; Li, Wen-Fei; Liu, Xu; Zhou, Guan-Qun; Sun, Ying; Kang, Tie-Bang; Zeng, Mu-Sheng; Liu, Na; Ma, Jun

    2015-01-01

    Latent membrane protein 1 (LMP1) is identified as the main transforming oncoprotein of Epstein-Barr virus (EBV). LMP1 is frequently expressed in a variety of EBV-associated cancers, including nasopharyngeal carcinoma (NPC), non-Hodgkin lymphoma (NHL), Hodgkin disease (HD), and gastric cancer (GC). However, due to conflicting results, the prognostic value of LMP1 expression on clinical outcomes in EBV-associated cancers remains unclear. We performed a meta-analysis on 32 studies with a total of 3752 patients to explore the association between LMP1 expression and overall survival (OS) in EBV-associated cancers. Overall, LMP1 expression was significantly associated with poorer OS (hazard ratio, HR = 1.51, 95% confidence interval, CI, 1.13–2.03), irrespective of cancer type. Further analyses showed that LMP1 expression correlated with poorer OS in NPC (HR = 2.48, 95% CI, 1.77–3.47) and NHL patients (HR = 1.83, 95% CI, 1.07–3.15), but not in HD patients (HR = 0.98, 95% CI, 0.60–1.62) or GC patients (HR = 0.70, 95% CI, 0.44–1.12). Subgroup analyses indicated that the age and geographical factors seemed to have an effect on the clinical outcomes of HD patients with positive LMP1 expression. In conclusion, LMP1 expression can be used as a prognostic biomarker in NPC, NHL, and certain HD patients. This data suggests that novel therapies targeting LMP1 may improve clinical outcomes for EBV-associated cancer patients. PMID:26336130

  6. The parvalbumin-positive interneurons in the mouse dentate gyrus express GABAA receptor subunits α1, β2, and δ along their extrasynaptic cell membrane.

    PubMed

    Milenkovic, I; Vasiljevic, M; Maurer, D; Höger, H; Klausberger, T; Sieghart, W

    2013-12-19

    Neuronal circuitries in the hippocampus are involved in navigation and memory and are controlled by major networks of GABAergic interneurons. Parvalbumin (PV)-expressing interneurons in the dentate gyrus (DG) are identified as fast-spiking cells, playing a crucial role in network oscillation and synchrony. The inhibitory modulation of these interneurons is thought to be mediated mainly through GABAA receptors, the major inhibitory neurotransmitter receptors in the brain. Here we show that all PV-positive interneurons in the granular/subgranular layer (GL/SGL) of the mouse DG express high levels of the GABAA receptor δ subunit. PV-containing interneurons in the hilus and the molecular layer, however, express the δ subunit to a lower extent. Only 8% of the somatostatin-containing interneurons express the δ subunit, whereas calbindin- or calretinin-containing interneurons in the DG seem not to express the GABAA receptor δ subunit at all. Hence, these cells receive a GABAergic control different from that of PV-containing interneurons in the GL/SGL. Experiments investigating a possible co-expression of GABAA receptor α1, α2, α3, α4, α5, β1, β2, β3, or γ2 subunits with PV and δ subunits indicated that α1 and β2 subunits are co-expressed with δ subunits along the extrasynaptic membranes of PV-interneurons. These results suggest a robust tonic GABAergic control of PV-containing interneurons in the GL/SGL of the DG via δ subunit-containing receptors. Our data are important for better understanding of the neuronal circuitries in the DG and the role of specific cell types under pathological conditions. PMID:24055402

  7. Effects of Low Amyloid-β (Aβ) Concentration on Aβ1–42 Oligomers Binding and GluN2B Membrane Expression

    PubMed Central

    Gilson, Virginie; Mbebi-Liegeois, Corinne; Sellal, François; de Barry, Jean

    2015-01-01

    Abstract Numerous studies have shown that amyloid-β (Aβ) modulate intracellular metabolic cascades and an intracellular Ca2+ homeostasis and a cell surface NMDA receptor expression alteration in Alzheimer’s disease (AD). However most of these findings have been obtained by using non-physiological Aβ concentrations. The present study deals with the effect of low Aβ concentrations on cellular homeostasis. We used nerve growth factor-differentiated PC12 cells and murine cortical neurons sequentially treated with low chronic monomeric or small oligomeric Aβ concentrations and high acute oligomeric Aβ concentrations to bring out a priming effect of chronic treatment on subsequently high Aβ concentrations-elicited cellular response. Both cell types indeed displayed an enhanced capacity to bind oligomeric Aβ after monomeric or small oligomeric Aβ application. Furthermore, the results show that monomeric Aβ1–42 application to the cells induces an increase of the Ca2+-response and of the membrane expression of the extrasynaptic subunit of the NMDA receptor GluN2B in PC12 cells, while the opposite effects were observed in cultured neurons. This suggests a sequential interaction of Aβ with the cellular plasma membrane involving monomers or small Aβ oligomers which would facilitate the binding of the deleterious high molecular Aβ oligomers. This mechanism would explain the slow progression of AD in the human nervous system and the deep gradient of neuronal death observed around the amyloid plaques in the nervous tissue. PMID:26401567

  8. A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

    NASA Astrophysics Data System (ADS)

    Kalra, Rajkumar S.; Wadhwa, Renu

    2015-02-01

    Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing & transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

  9. A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

    SciTech Connect

    Kalra, Rajkumar S. Wadhwa, Renu

    2015-02-27

    Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

  10. Levetiracetam Differentially Alters CD95 Expression of Neuronal Cells and the Mitochondrial Membrane Potential of Immune and Neuronal Cells in vitro.

    PubMed

    Rogers, Susannah K; Shapiro, Lee A; Tobin, Richard P; Tow, Benjamin; Zuzek, Aleksej; Mukherjee, Sanjib; Newell-Rogers, M Karen

    2014-01-01

    Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s) of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side-effects. The current study examined the effects of levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if levetiracetam alters the expression of immune receptor-ligand pairs. The results show that levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action. PMID:24600432

  11. MT4-(MMP17) and MT6-MMP (MMP25), A unique set of membrane-anchored matrix metalloproteinases: properties and expression in cancer

    PubMed Central

    Sohail, Anjum; Sun, Qing; Zhao, Huiren; Bernardo, M. Margarida; Cho, Jin-Ah

    2014-01-01

    The process of cancer progression involves the action of multiple proteolytic systems, among which the family of matrix metalloproteinases (MMPs) play a pivotal role. The MMPs evolved to accomplish their proteolytic tasks in multiple cellular and tissue microenvironments including lipid rafts by incorporation and deletions of specific structural domains. The membrane type-MMPs (MT-MMPs) incorporated membrane anchoring domains that display these proteases at the cell surface, and thus they are optimal pericellular proteolytic machines. Two members of the MT-MMP subfamily, MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP), are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor, which confers these enzymes a unique set of regulatory and functional mechanisms that separates them from the rest of the MMP family. Discovered almost a decade ago, the body of work on GPI-MT-MMPs today is still surprisingly limited when compared to other MT-MMPs. However, new evidence shows that the GPI-MT-MMPs are highly expressed in human cancer, where they are associated with progression. Accumulating biochemical and functional evidence also highlights their distinct properties. In this review, we summarize the structural, biochemical, and biological properties of GPI-MT-MMPs and present an overview of their expression and role in cancer. We further discuss the potential implications of GPI-anchoring for enzyme function. Finally, we comment on the new scientific challenges that lie ahead to better understand the function and role in cancer of these intriguing but yet unique MMPs. PMID:18286233

  12. Selective upregulation of the expression of plasma membrane calcium ATPase isoforms upon differentiation and 1,25(OH)2D3-vitamin treatment of colon cancer cells.

    PubMed

    Ribiczey, Polett; Papp, Béla; Homolya, László; Enyedi, Ágnes; Kovács, Tünde

    2015-08-14

    We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca(2+) absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca(2+) absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca(2+) transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca(2+) signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3

  13. Membranous expressions of Lewis y and CAM-DR-related markers are independent factors of chemotherapy resistance and poor prognosis in epithelial ovarian cancer

    PubMed Central

    Zhu, Lian-Cheng; Gao, Jian; Hu, Zhen-Hua; Schwab, Carlton L; Zhuang, Hui-Yu; Tan, Ming-Zi; Yan, Li-Mei; Liu, Juan-Juan; Zhang, Dan-Ye; Lin, Bei

    2015-01-01

    Background: Chemotherapy resistance is a common problem faced by patients diagnosed with epithelial ovarian cancer (EOC). Currently there are no specific or sensitive clinical biomarkers that maybe implemented to identify chemotherapy resistance and give insight to prognosis. The aim of this study is to investigate the roles of Lewis y antigen and the markers associated with cell-adhesion-mediated drug resistance (CAM-DR) in patients with EOC. Methods: 92 EOC patients who were treated with systemic chemotherapy after cytoreductive surgery were included in this analysis. Patients were divided into two groups, chemotherapy sensitive (n = 56) and resistant (n = 36). Immunohistochemical (IHC) staining for Lewis y and CAM-DR-related cell surface proteins including CD44, CD147, HE4 (Human epididymis protein 4), integrin α5, β1, αv and β3 were conducted on tissues collected during primary debulking surgery. Using multivariate logistic regressions, IHC results were compared to clinical variables and chemotherapy resistance to determine possible correlations. The relationships between IHC expression and progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan–Meier method and Cox regression analysis. Results: Membranous expression of Lewis y and all these CAM-DR-related markers were significantly higher in the resistant group than that of the sensitive group (all P < 0.01). Multivariate regression analysis revealed that high expression of Lewis y, CD44, HE4, integrin α5 and β1 as well as advanced FIGO stage were independent risk factors for chemotherapy resistance (all P < 0.05). Advanced FIGO stage, lymph node metastasis and high expression of Lewis y, CD44, CD147, HE4, integrin α5, β1 were associated with a shorter PFS and OS (all P < 0.05). Moreover, multivariate COX analysis demonstrated that the following variates were independent predictors of worse PFS and OS survival: late FIGO stage (P = 0.013, 0.049), high expressions of Lewis

  14. THE C2 OXIDATIVE PHOTOSYNTHETIC CARBON CYCLE.

    PubMed

    Tolbert, N. E.

    1997-06-01

    The C2 oxidative photosynthetic carbon cycle plus the C3 reductive photosynthetic carbon cycle coexist. Both are initiated by Rubisco, use about equal amounts of energy, must regenerate RuBP, and result in exchanges of CO2 and O2 to establish rates of net photosynthesis, CO2 and O2 compensation points, and the ratio of CO2 and O2 in the atmosphere. These concepts evolved from research on O2 inhibition, glycolate metabolism, leaf peroxisomes, photorespiration, 18O2/16O2 exchange, CO2 concentrating processes, and a requirement for the oxygenase activity of Rubisco. Nearly 80 years of research on these topics are unified under the one process of photosynthetic carbon metabolism and its self-regulation. PMID:15012254

  15. BOREAS TE-10 Photosynthetic Response Data

    NASA Technical Reports Server (NTRS)

    Hall, Forrest G. (Editor); Papagno, Andrea (Editor); Middleton, Elizabeth; Sullivan, Joseph

    2000-01-01

    The Boreal Ecosystem-Atmospheric Study (BOREAS) TE-10 (Terrestrial Ecology) team collected several data sets in support of its efforts to characterize and interpret information on the gas exchange, reflectance, transmittance, chlorophyll content, carbon content, hydrogen content, nitrogen content, and photosynthetic response of boreal vegetation. This data set contains measurements of quantitative parameters and leaf photosynthetic response to increases in light conducted in the SSA during the growing seasons of 1994 and 1996 using an oxygen electrode system. Leaf photosynthetic responses were not collected in 1996. The data are stored in tabular ASCII files. The data files are available on a CD-ROM (see document number 20010000884), or from the Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC).

  16. Photosynthetic complex stoichiometry dynamics in higher plants: environmental acclimation and photosynthetic flux control

    PubMed Central

    Schöttler, Mark A.; Tóth, Szilvia Z.

    2014-01-01

    The composition of the photosynthetic apparatus of higher plants is dynamically adjusted to long-term changes in environmental conditions such as growth light intensity and light quality, and to changing metabolic demands for ATP and NADPH imposed by stresses and leaf aging. By changing photosynthetic complex stoichiometry, a long-term imbalance between the photosynthetic production of ATP and NADPH and their metabolic consumption is avoided, and cytotoxic side reactions are minimized. Otherwise, an excess capacity of the light reactions, relative to the demands of primary metabolism, could result in a disturbance of cellular redox homeostasis and an increased production of reactive oxygen species, leading to the destruction of the photosynthetic apparatus and the initiation of cell death programs. In this review, changes of the abundances of the different constituents of the photosynthetic apparatus in response to environmental conditions and during leaf ontogenesis are summarized. The contributions of the different photosynthetic complexes to photosynthetic flux control and the regulation of electron transport are discussed. PMID:24860580

  17. Design criteria for optimal photosynthetic energy conversion

    NASA Astrophysics Data System (ADS)

    Fingerhut, Benjamin P.; Zinth, Wolfgang; de Vivie-Riedle, Regina

    2008-12-01

    Photochemical solar energy conversion is considered as an alternative of clean energy. For future light converting nano-machines photosynthetic reaction centers are used as prototypes optimized during evolution. We introduce a reaction scheme for global optimization and simulate the ultrafast charge separation in photochemical energy conversion. Multiple molecular charge carriers are involved in this process and are linked by Marcus-type electron transfer. In combination with evolutionary algorithms, we unravel the biological strategies for high quantum efficiency in photosynthetic reaction centers and extend these concepts to the design of artificial photochemical devices for energy conversion.

  18. Enhanced practical photosynthetic CO2 mitigation

    DOEpatents

    Bayless, David J.; Vis-Chiasson, Morgan L.; Kremer, Gregory G.

    2003-12-23

    This process is unique in photosynthetic carbon sequestration. An on-site biological sequestration system directly decreases the concentration of carbon-containing compounds in the emissions of fossil generation units. In this process, photosynthetic microbes are attached to a growth surface arranged in a containment chamber that is lit by solar photons. A harvesting system ensures maximum organism growth and rate of CO.sub.2 uptake. Soluble carbon and nitrogen concentrations delivered to the cyanobacteria are enhanced, further increasing growth rate and carbon utilization.

  19. Expression of a soluble form of iodotyrosine deiodinase for active site characterization by engineering the native membrane protein from Mus musculus

    SciTech Connect

    Buss, Jennifer M.; McTamney, Patrick M.; Rokita, Steven E.

    2012-06-27

    Reductive deiodination is critical for thyroid function and represents an unusual exception to the more common oxidative and hydrolytic mechanisms of dehalogenation in mammals. Studies on the reductive processes have been limited by a lack of convenient methods for heterologous expression of the appropriate proteins in large scale. The enzyme responsible for iodide salvage in the thyroid, iodotyrosine deodinase, is now readily generated after engineering its gene from Mus musculus. High expression of a truncated derivative lacking the membrane domain at its N-terminal was observed in Sf9 cells, whereas expression in Pichia pastoris remained low despite codon optimization. Ultimately, the desired expression in Escherichia coli was achieved after replacing the two conserved Cys residues of the deiodinase with Ala and fusing the resulting protein to thioredoxin. This final construct provided abundant enzyme for crystallography and mutagenesis. Utility of the E. coli system was demonstrated by examining a set of active site residues critical for binding to the zwitterionic portion of substrate.

  20. [Wogonin inhibits IGF-1-stimulated cell growth and estrogen receptor α expression in breast adenocarcinoma cell and angiogenesis of chick chorioallantoic membrane].

    PubMed

    Ma, Xing; Xie, Kun-Peng; Shang, Fei; Huo, Hong-Nan; Wang, Li-Meng; Xie, Ming-Jie

    2012-04-25

    The aim of the present study was to investigate the involvements of insulin-like growth factor-1 (IGF-1) and estrogen receptor α (ERα) in the inhibitory effect of wogonin on the breast adenocarcinoma growth. Moreover, the effect of wogonin on the angiogenesis of chick chorioallantoic membrane (CAM) was also investigated. MCF-7 cells (human breast adenocarcinoma cell line) were subjected to several drugs, including IGF-1, wogonin and ER inhibitor ICI182780, alone or in combination. MTT assay was used to detect breast cancer proliferation. Western blot was used to analyze ERα and p-Akt expression levels. CAM models prepared from 6-day chicken eggs were employed to evaluate angiogenesis inhibition. The results showed wogonin and ICI182780 both exhibited a potent ability to blunt IGF-1-stimulated MCF-7 cell growth. Either of wogonin and ICI182780 significantly inhibited ERα and p-Akt expressions in IGF-1-treated cells. The inhibitory effect of wogonin showed no difference from that of ICI182780 on IGF-1-stimulated expressions of ERα and p-Akt. Meanwhile, wogonin at different concentrations showed significant inhibitory effect on CAM angiogenesis. These results suggest the inhibitory effect of wogonin on breast adenocarcinoma growth via inhibiting IGF-1-mediated PI3K-Akt pathway and regulating ERα expression. Furthermore, wogonin has a strong anti-angiogenic effect on CAM model. PMID:22513472

  1. Characterization of AgMaT2, a Plasma Membrane Mannitol Transporter from Celery, Expressed in Phloem Cells, Including Phloem Parenchyma Cells[OA

    PubMed Central

    Juchaux-Cachau, Marjorie; Landouar-Arsivaud, Lucie; Pichaut, Jean-Philippe; Campion, Claire; Porcheron, Benoit; Jeauffre, Julien; Noiraud-Romy, Nathalie; Simoneau, Philippe; Maurousset, Laurence; Lemoine, Rémi

    2007-01-01

    A second mannitol transporter, AgMaT2, was identified in celery (Apium graveolens L. var. dulce), a species that synthesizes and transports mannitol. This transporter was successfully expressed in two different heterologous expression systems: baker's yeast (Saccharomyces cerevisiae) cells and tobacco (Nicotiana tabacum) plants (a non-mannitol-producing species). Data indicated that AgMaT2 works as an H+/mannitol cotransporter with a weak selectivity toward other polyol molecules. When expressed in tobacco, AgMaT2 decreased the sensitivity to the mannitol-secreting pathogenic fungi Alternaria longipes, suggesting a role for polyol transporters in defense mechanisms. In celery, in situ hybridization showed that AgMaT2 was expressed in the phloem of leaflets, petioles from young and mature leaves, floral stems, and roots. In the phloem of petioles and leaflets, AgMaT2, as localized with specific antibodies, was present in the plasma membrane of three ontologically related cell types: sieve elements, companion cells, and phloem parenchyma cells. These new data are discussed in relation to the physiological role of AgMaT2 in regulating mannitol fluxes in celery petioles. PMID:17631523

  2. A comparative study of the photosynthetic capacity in two green tide macroalgae using chlorophyll fluorescence.

    PubMed

    Wang, Ying; Qu, Tongfei; Zhao, Xinyu; Tang, Xianghai; Xiao, Hui; Tang, Xuexi

    2016-01-01

    Green tides have occurred in the Yellow Sea, China, every year from 2007 to 2015. The free-floating Ulva prolifera (Müller) J. Agardh was the causative macroalgal species. The co-occurring, attached U. intestinalis was also observed. Photosynthetic capacities were determined using chlorophyll fluorescence in situ and after 7 days lab acclimation, and a significant differences were noted. Pigment composition showed no obvious differences, but concentrations varied significantly, especially chlorophyll b in U. prolifera two times increase was observed after acclimation. The optimal photochemical efficiency of PS II (Fv/Fm) was significantly higher in U. prolifera. Photosynthetic rate (α), maximum relative electron transport rate (rETRmax), and minimum saturating irradiance (Ek), obtained from rapid light response curves (RLCs), showed almost the same photosynthetic physiological status as Fv/Fm. Quenching coefficients and low temperature (77 K) chlorophyll fluorescence emission spectra of thylakoid membranes analysis showed U. prolifera has a better recovery activity and plasticity of PSII than U. intestinalis. Furthermore, energy dissipation via non-photochemical quenching (NPQ) and state transitions showed efficacious photoprotection solution especially in U. prolifera suffered from the severe stresses. Results in the present study suggested that U. prolifera's higher photosynthetic capacity would contribute to its free-floating proliferation, and efficacious photoprotection in addition to favorable oceanographic conditions and high nutrient levels support its growth and aggregation. PMID:27386261

  3. Light stress photodynamics of chlorophyll-binding proteins in Arabidopsis thaliana thylakoid membranes revealed by high-resolution mass spectrometric studies.

    PubMed

    Galetskiy, D N; Lohscheider, J N; Kononikhin, A S; Kharybin, O N; Popov, I A; Adamska, I; Nikolaev, E N

    2011-01-01

    In higher plants the light energy is captured by the photosynthetic pigments that are bound to photosystem I and II and their light-harvesting complex (LHC) subunits. In this study, we examined the photodynamic changes within chlorophyll-protein complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to low light and subsequently exposed to light stress. Chlorophyll-protein complexes were isolated using sucrose density gradient centrifugation and blue-native polyacrylamid gel electrophoresis (BN-PAGE). Proteome analysis was performed using SDS-PAGE, HPLC and high resolution mass spectrometry. We identified several rarely expressed and stress-induced chlorophyll-binding proteins, showed changes in localization of early light-induced protein family and LHC protein family members between different photosynthetic complexes and assembled/disassembled subcomplexes under light stress conditions and discuss their role in a variety of light stress-related processes. PMID:21460887

  4. Lack of correlation between membrane CD30 expression and cytokine secretion pattern in allergen-primed naive cord blood T-cell lines and clones.

    PubMed

    Spinozzi, F; Agea, E; Piattoni, S; Falini, B; Grignani, F; Bertotto, A

    1997-04-01

    Various surface molecules are expressed by activated T cells. Among them, the CD30 antigen has been proposed as a reproducible marker that identifies a subset of differentiated and/or activated T lymphocytes that produce T helper (Th)-2-type cytokines, i.e. interleukin (IL)-4 and IL-5. However, because CD30 has mainly been detected on established T-cell clones, it is still unclear whether a priming allergen and/or cytokine can induce its membrane expression on naive T cells, perhaps in parallel with the up-regulation of other relevant activation markers, such as CD25, HLA-DR and L-selectin. It is also unknown whether proper allergen stimulation affects the cytokine secretion pattern by CD30+ T-cell clones derived from antigen-unprimed (naive) T lymphocytes. More information on these questions was sought by adopting a model that used cord blood as a source of virgin T cells and exposing them to native cypress allergen or cytokine (IL-2 or IL-4) stimulation, as well as to conventional polyclonal activators such as PHA or anti-CD3. Peripheral blood MC from four adult cypress-sensitive patients was also assayed and used as controls for all culture experiments. Freshly isolated cord and adult T cells did not express the CD30 antigen on their membrane. Many of the stimulating agents tested were able to up-regulate the expression of CD30. However, despite high expression of this molecule, cloned allergen-specific cord CD4+ T lymphocytes were unable to produce IFN-gamma and/or IL-4. In contrast, they retained the capability to produce IL-2. Thus, expression of the CD30 antigen on virgin T cells does not correlate with a polarized model of T helper (Th)-1 or Th-2 cytokine-producing cells, suggesting that these types of lymphokine-secreting lymphocytes are not a paradigmatic example of T-cell subpopulations that display stable phenotypical features. PMID:9105430

  5. iTRAQ-based analysis of developmental dynamics in the soybean leaf proteome reveals pathways associated with leaf