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Sample records for photosynthetic membrane expression

  1. 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...

  2. Fluctuating Two-State Light Harvesting in a Photosynthetic Membrane

    SciTech Connect

    Pan, Duohai; Hu, Dehong; Liu, Ruchuan; Zeng, Xiaohua; Kaplan, Samuel; Lu, H. Peter

    2007-06-28

    How light is converted into chemical energy in a natural photosynthetic system is of great interest in energy sciences. Using single-molecule and single-vesicle fluorescence spectroscopy and imaging, we have observed fluctuating inter-molecular protein energy transfers in the photosynthetic membranes of R. sphaeroides. Our results suggest that there are dynamic coupled and non-coupled states in the light-harvesting protein assembly.

  3. 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.

  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. Photosynthetic Membrane System in Anacystis nidulans

    PubMed Central

    Allen, Mary Mennes

    1968-01-01

    Cultures of Anacystis nidulans were grown under conditions of varying light intensity and temperature. Changes in pigment content were compared with changes in the fine structure of these cells. Pigment concentration and lamellar content varied inversely with the light intensity in cells grown with 100 and 1,000 foot candles of fluorescent light. Estimations of the relative area and volume of lamellae in cells showed that the amount of double membrane was directly proportional to the chlorophyll content of whole cells. Continuity of double membranes with cytoplasmic membrane was observed. Images PMID:5732512

  6. Photosynthetic Membranes of Porphyridium cruentum1

    PubMed Central

    Redlinger, Thomas; Gantt, Elisabeth

    1983-01-01

    Three chlorophyll-protein complexes (CP I, CP III, CP IV) were electrophoretically separated from thylakoids of the eukaryotic red alga Porphyridium cruentum. CP I contained the primary photochemical reaction center of photosystem I as judged by its light-induced reversible absorbance change at 700 nanometers, by its fluorescence emission maximum at 720 nanometers (−196°C), and by the molecular weight of its apoprotein (68,000 daltons). CP III and CP IV appeared to belong with photosystem II as suggested by the absence of light-reversible absorbance at 700 nanometers, by their fluorescence maximum at 690 nanometers (−196°C), and by the presence of a chlorophyll-binding polypeptide with a molecular weight of about 52,000 daltons. CP IV when completely denatured had two additional polypeptides of about 40,000 and 48,000 daltons. All three chlorophyll-protein complexes contained carotenoids: the chlorophyll/carotenoid molar ratio of 15:1 for CP I, and 20:1 for CP III and CP IV. The thylakoid membranes of P. cruentum contained four cytochromes, detected by heme-dependent peroxidase activity, but there was no observed association with the electrophoretically separated chlorophyll-protein complexes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 7 PMID:16663181

  7. Probing the dynamics of photosynthetic membranes with fluorescence recovery after photobleaching.

    PubMed

    Mullineaux, Conrad W; Sarcina, Mary

    2002-06-01

    In the past few years, there has been remarkable progress in knowledge of the structures and organization of the protein complexes of photosynthetic membranes. However, static structures do not tell the whole story. Photosynthetic membranes, like other biological membranes, are dynamic systems. Recent technological advances are making it increasingly easy to probe the dynamics of photosynthetic membranes using fluorescence recovery after photobleaching. Here we explain the potential and the limitations of the technique.

  8. 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

    2015-11-03

    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.

  9. Elementary Energy Transfer Pathways in Allochromatium vinosum Photosynthetic Membranes

    PubMed Central

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

    2015-01-01

    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. Architectural switch in plant photosynthetic membranes induced by light stress.

    PubMed

    Herbstová, Miroslava; Tietz, Stefanie; Kinzel, Christopher; Turkina, Maria V; Kirchhoff, Helmut

    2012-12-04

    Unavoidable side reactions of photosynthetic energy conversion can damage the water-splitting photosystem II (PSII) holocomplex embedded in the thylakoid membrane system inside chloroplasts. Plant survival is crucially dependent on an efficient molecular repair of damaged PSII realized by a multistep repair cycle. The PSII repair cycle requires a brisk lateral protein traffic between stacked grana thylakoids and unstacked stroma lamellae that is challenged by the tight stacking and low protein mobility in grana. We demonstrated that high light stress induced two main structural changes that work synergistically to improve the accessibility between damaged PSII in grana and its repair machinery in stroma lamellae: lateral shrinkage of grana diameter and increased protein mobility in grana thylakoids. It follows that high light stress triggers an architectural switch of the thylakoid network that is advantageous for swift protein repair. Studies of the thylakoid kinase mutant stn8 and the double mutant stn7/8 demonstrate the central role of protein phosphorylation for the structural alterations. These findings are based on the elaboration of mathematical tools for analyzing confocal laser-scanning microscopic images to study changes in the sophisticated thylakoid architecture in intact protoplasts.

  11. Revealing linear aggregates of light harvesting antenna proteins in photosynthetic membranes.

    PubMed

    He, Yufan; Zeng, Xiaohua; Mukherjee, Saptarshi; Rajapaksha, Suneth; Kaplan, Samuel; Lu, H Peter

    2010-01-05

    How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions by using atomic force microscopy (AFM) imaging and spectroscopic analysis. Our results suggest that the light harvesting protein, LH2, can exist as linear aggregates of 4 +/- 2 proteins in the photosynthetic membranes and that the protein distributions are highly heterogeneous. In the photosynthetic membranes examined in our measurements, the ratio of the aggregated to the nonaggregated LH2 proteins is about 3:1 to 5:1 depending on the intensity of the illumination used during sample incubation and on the bacterial species. AFM images further identify that the LH2 proteins in the linear aggregates are monotonically tilted at an angle 4 +/- 2 degrees from the plane of the photosynthetic membranes. The aggregates result in red-shifted absorption and emission spectra that are measured using various mutant membranes, including an LH2 knockout, LH1 knockout, and LH2 at different population densities. Measuring the fluorescence lifetimes of purified LH2 and LH2 in membranes, we have observed that the LH2 proteins in membranes exhibit biexponential lifetime decays whereas the purified LH2 proteins gave single exponential lifetime decays. We attribute that the two lifetime components originate from the existence of both aggregated and nonaggregated LH2 proteins in the photosynthetic membranes.

  12. 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.

  13. 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

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

    PubMed

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

    2015-08-28

    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.

  15. Membrane development in purple photosynthetic bacteria in response to alterations in light intensity and oxygen tension.

    PubMed

    Niederman, Robert A

    2013-10-01

    Studies on membrane development in purple bacteria during adaptation to alterations in light intensity and oxygen tension are reviewed. Anoxygenic phototrophic such as the purple α-proteobacterium Rhodobacter sphaeroides have served as simple, dynamic, and experimentally accessible model organisms for studies of the photosynthetic apparatus. A major landmark in photosynthesis research, which dramatically illustrates this point, was provided by the determination of the X-ray structure of the reaction center (RC) in Blastochloris viridis (Deisenhofer and Michel, EMBO J 8:2149-2170, 1989), once it was realized that this represented the general structure for the photosystem II RC present in all oxygenic phototrophs. This seminal advance, together with a considerable body of subsequent research on the light-harvesting (LH) and electron transfer components of the photosynthetic apparatus has provided a firm basis for the current understanding of how phototrophs acclimate to alterations in light intensity and quality. Oxygenic phototrophs adapt to these changes by extensive thylakoid membrane remodeling, which results in a dramatic supramolecular reordering to assure that an appropriate flow of quinone redox species occurs within the membrane bilayer for efficient and rapid electron transfer. Despite the high level of photosynthetic unit organization in Rba. sphaeroides as observed by atomic force microscopy (AFM), fluorescence induction/relaxation measurements have demonstrated that the addition of the peripheral LH2 antenna complex in cells adapting to low-intensity illumination results in a slowing of the rate of electron transfer turnover by the RC of up to an order of magnitude. This is ascribed to constraints in quinone redox species diffusion between the RC and cytochrome bc1 complexes arising from the increased packing density as the intracytoplasmic membrane (ICM) bilayer becomes crowded with LH2 rings. In addition to downshifts in light intensity as a paradigm

  16. Inelastic neutron scattering study of light-induced dynamics of a photosynthetic membrane system

    SciTech Connect

    Furrer, A.; Stoeckli, A.

    2010-01-15

    Inelastic neutron scattering was employed to study photoeffects on the molecular dynamics of membranes of the photosynthetic bacterium Rhodopseudomonas viridis. The main photoactive parts of this biomolecular system are the chlorophyll molecules whose dynamics were found to be affected under illumination by visible light in a twofold manner. First, vibrational modes are excited at energies of 12(2) and 88(21) cm{sup -1}. Second, a partial 'freezing' of rotational modes is observed at energies of 1.2(3) and 2.9(5) cm{sup -1}. These results are attributed to a possible coupling between molecular motions and particular mechanisms in the photosynthetic process.

  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. Biogenesis of cytochrome b6 in photosynthetic membranes

    PubMed Central

    Saint-Marcoux, Denis; Wollman, Francis-André

    2009-01-01

    In chloroplasts, binding of a c′-heme to cytochrome b6 on the stromal side of the thylakoid membranes requires a specific mechanism distinct from the one at work for c-heme binding to cytochromes f and c6 on the lumenal side of membranes. Here, we show that the major protein components of this pathway, the CCBs, are bona fide transmembrane proteins. We demonstrate their association in a series of hetero-oligomeric complexes, some of which interact transiently with cytochrome b6 in the process of heme delivery to the apoprotein. In addition, we provide preliminary evidence for functional assembly of cytochrome b6f complexes even in the absence of c′-heme binding to cytochrome b6. Finally, we present a sequential model for apo- to holo-cytochrome b6 maturation integrated within the assembly pathway of b6f complexes in the thylakoid membranes. PMID:19564403

  19. Biogenesis of cytochrome b6 in photosynthetic membranes.

    PubMed

    Saint-Marcoux, Denis; Wollman, Francis-André; de Vitry, Catherine

    2009-06-29

    In chloroplasts, binding of a c'-heme to cytochrome b(6) on the stromal side of the thylakoid membranes requires a specific mechanism distinct from the one at work for c-heme binding to cytochromes f and c(6) on the lumenal side of membranes. Here, we show that the major protein components of this pathway, the CCBs, are bona fide transmembrane proteins. We demonstrate their association in a series of hetero-oligomeric complexes, some of which interact transiently with cytochrome b(6) in the process of heme delivery to the apoprotein. In addition, we provide preliminary evidence for functional assembly of cytochrome b(6)f complexes even in the absence of c'-heme binding to cytochrome b(6). Finally, we present a sequential model for apo- to holo-cytochrome b(6) maturation integrated within the assembly pathway of b(6)f complexes in the thylakoid membranes.

  20. 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.

  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. 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.

  3. 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.

  4. 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.

  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. Energy transfer dynamics in an RC-LH1-PufX tubular photosynthetic membrane

    NASA Astrophysics Data System (ADS)

    Hsin, J.; Strümpfer, J.; Şener, M.; Qian, P.; Hunter, C. N.; Schulten, K.

    2010-08-01

    Light absorption and the subsequent transfer of excitation energy are the first two steps in the photosynthetic process, carried out by protein-bound pigments, mainly bacteriochlorophylls (BChls), in photosynthetic bacteria. BChls are anchored in light-harvesting (LH) complexes, such as light-harvesting complex I (LH1), which directly associates with the reaction center (RC), forming the RC-LH1 core complex. In Rhodobacter sphaeroides, RC-LH1 core complexes contain an additional protein, PufX, and assemble into dimeric RC-LH1-PufX core complexes. In the absence of LH complex II (LH2), the former complexes can aggregate into a helically ordered tubular photosynthetic membrane. We have examined the excitation transfer dynamics in a single RC-LH1-PufX core complex dimer using the hierarchical equations of motion for dissipative quantum dynamics that accurately, yet in a computationally costly manner, treat the coupling between BChls and their protein environment. A widely employed description, the generalized Förster (GF) theory, was also used to calculate the transfer rates of the same excitonic system in order to verify the accuracy of this computationally cheap method. Additionally, in light of the structural uncertainties in the Rba. sphaeroides RC-LH1-PufX core complex, geometrical alterations were introduced into the BChl organization. It is shown that the energy transfer dynamics are not affected by the considered changes in the BChl organization and that the GF theory provides accurate transfer rates. An all-atom model for a tubular photosynthetic membrane is then constructed on the basis of electron microscopy data, and the overall energy transfer properties of this membrane are computed.

  7. Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle.

    PubMed

    Sener, Melih K; Olsen, John D; Hunter, C Neil; Schulten, Klaus

    2007-10-02

    The photosynthetic unit (PSU) of purple photosynthetic bacteria consists of a network of bacteriochlorophyll-protein complexes that absorb solar energy for eventual conversion to ATP. Because of its remarkable simplicity, the PSU can serve as a prototype for studies of cellular organelles. In the purple bacterium Rhodobacter sphaeroides the PSU forms spherical invaginations of the inner membrane, approximately 70 nm in diameter, composed mostly of light-harvesting complexes, LH1 and LH2, and reaction centers (RCs). Atomic force microscopy studies of the intracytoplasmic membrane have revealed the overall spatial organization of the PSU. In the present study these atomic force microscopy data were used to construct three-dimensional models of an entire membrane vesicle at the atomic level by using the known structure of the LH2 complex and a structural model of the dimeric RC-LH1 complex. Two models depict vesicles consisting of 9 or 18 dimeric RC-LH1 complexes and 144 or 101 LH2 complexes, representing a total of 3,879 or 4,464 bacteriochlorophylls, respectively. The in silico reconstructions permit a detailed description of light absorption and electronic excitation migration, including computation of a 50-ps excitation lifetime and a 95% quantum efficiency for one of the model membranes, and demonstration of excitation sharing within the closely packed RC-LH1 dimer arrays.

  8. Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria.

    PubMed

    Bashford, C L; Chance, B; Prince, R C

    1979-01-11

    The reponses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the 'light-minus-dark' difference spectrum of the chromatophores. The oxonols appear to respond to the light-induced 'energization' by shifting their absorption maxima. In the presence of K+, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes, respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift. The dye response has an apparent second-order rate constant of approx. 2 . 10(6) M-1 . s-1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not possess intrinsic probes of potential.

  9. 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.

  10. Still Acting Green: Continued Expression of Photosynthetic Genes in the Heterotrophic Dinoflagellate Pfiesteria piscicida (Peridiniales, Alveolata)

    PubMed Central

    Kim, Gwang Hoon; Jeong, Hae Jin; Yoo, Yeong Du; Kim, Sunju; Han, Ji Hee; Han, Jong Won; Zuccarello, Giuseppe C.

    2013-01-01

    The loss of photosynthetic function should lead to the cessation of expression and finally loss of photosynthetic genes in the new heterotroph. Dinoflagellates are known to have lost their photosynthetic ability several times. Dinoflagellates have also acquired photosynthesis from other organisms, either on a long-term basis or as “kleptoplastids” multiple times. The fate of photosynthetic gene expression in heterotrophs can be informative into evolution of gene expression patterns after functional loss, and the dinoflagellates ability to acquire new photosynthetic function through additional endosymbiosis. To explore this we analyzed a large-scale EST database consisting of 151,091 unique sequences (29,170 contigs, 120,921 singletons) obtained from 454 pyrosequencing of the heterotrophic dinoflagellate Pfiesteria piscicida. About 597 contigs from P. piscicida showed significant homology (E-value photosynthetic function. Most of the genes involved in the Calvin-Benson cycle were found, genes of the light-dependent reaction were also identified. Also genes of associated pathways including the chorismate pathway and genes involved in starch metabolism were discovered. BLAST searches and phylogenetic analysis suggest that these plastid-associated genes originated from several different photosynthetic ancestors. The Calvin-Benson cycle genes are mostly associated with genes derived from the secondary plastids of peridinin-containing dinoflagellates, while the light-harvesting genes are derived from diatoms, or diatoms that are tertiary plastids in other dinoflagellates. The continued expression of many genes involved in photosynthetic pathways indicates that the loss of transcriptional regulation may occur well after plastid loss and could explain the organism's ability to “capture” new plastids (i.e. different secondary endosymbiosis or tertiary symbioses) to renew photosynthetic function. PMID:23874554

  11. Still acting green: continued expression of photosynthetic genes in the heterotrophic Dinoflagellate Pfiesteria piscicida (Peridiniales, Alveolata).

    PubMed

    Kim, Gwang Hoon; Jeong, Hae Jin; Yoo, Yeong Du; Kim, Sunju; Han, Ji Hee; Han, Jong Won; Zuccarello, Giuseppe C

    2013-01-01

    The loss of photosynthetic function should lead to the cessation of expression and finally loss of photosynthetic genes in the new heterotroph. Dinoflagellates are known to have lost their photosynthetic ability several times. Dinoflagellates have also acquired photosynthesis from other organisms, either on a long-term basis or as "kleptoplastids" multiple times. The fate of photosynthetic gene expression in heterotrophs can be informative into evolution of gene expression patterns after functional loss, and the dinoflagellates ability to acquire new photosynthetic function through additional endosymbiosis. To explore this we analyzed a large-scale EST database consisting of 151,091 unique sequences (29,170 contigs, 120,921 singletons) obtained from 454 pyrosequencing of the heterotrophic dinoflagellate Pfiesteria piscicida. About 597 contigs from P. piscicida showed significant homology (E-value photosynthetic function. Most of the genes involved in the Calvin-Benson cycle were found, genes of the light-dependent reaction were also identified. Also genes of associated pathways including the chorismate pathway and genes involved in starch metabolism were discovered. BLAST searches and phylogenetic analysis suggest that these plastid-associated genes originated from several different photosynthetic ancestors. The Calvin-Benson cycle genes are mostly associated with genes derived from the secondary plastids of peridinin-containing dinoflagellates, while the light-harvesting genes are derived from diatoms, or diatoms that are tertiary plastids in other dinoflagellates. The continued expression of many genes involved in photosynthetic pathways indicates that the loss of transcriptional regulation may occur well after plastid loss and could explain the organism's ability to "capture" new plastids (i.e. different secondary endosymbiosis or tertiary symbioses) to renew photosynthetic function.

  12. Enhancing soybean photosynthetic CO2 assimilation using a cyanobacterial membrane protein, ictB.

    PubMed

    Hay, William T; Bihmidine, Saadia; Mutlu, Nedim; Hoang, Khang Le; Awada, Tala; Weeks, Donald P; Clemente, Tom E; Long, Stephen P

    2017-02-16

    Soybean C3 photosynthesis can suffer a severe loss in efficiency due to photorespiration and the lack of a carbon concentrating mechanism (CCM) such as those present in other plant species or cyanobacteria. Transgenic soybean (Glycine max cv. Thorne) plants constitutively expressing cyanobacterial ictB (inorganic carbon transporter B) gene were generated using Agrobacterium-mediated transformation. Although more recent data suggest that ictB does not actively transport HCO3-/CO2, there is nevertheless mounting evidence that transformation with this gene can increase higher plant photosynthesis. The hypothesis that expression of the ictB gene would improve photosynthesis, biomass production and seed yield in soybean was tested, in two independent replicated greenhouse and field trials. Results showed significant increases in photosynthetic CO2 uptake (Anet) and dry mass in transgenic relative to wild type (WT) control plants in both the greenhouse and field trials. Transgenic plants also showed increased photosynthetic rates and biomass production during a drought mimic study. The findings presented herein demonstrate that ictB, as a single-gene, contributes to enhancement in various yield parameters in a major commodity crop and point to the significant role that biotechnological approaches to increasing photosynthetic efficiency can play in helping to meet increased global demands for food.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24561561

  14. Electroporation of the photosynthetic membrane: structural changes in protein and lipid-protein domains.

    PubMed Central

    Rosemberg, Y; Rotenberg, M; Korenstein, R

    1994-01-01

    A biological membrane undergoes a reversible permeability increase through structural changes in the lipid domain when exposed to high external electric fields. The present study shows the occurrence of electric field-induced changes in the conductance of the proton channel of the H(+)-ATPase as well as electric field-induced structural changes in the lipid-protein domain of photosystem (PS) II in the photosynthetic membrane. The study was carried out by analyzing the electric field-stimulated delayed luminescence (EPL), which originates from charge recombination in the protein complexes of PS I and II of photosynthetic vesicles. We established that a small fraction of the total electric field-induced conductance change was abolished by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the H(+)-ATPase. This reversible electric field-induced conductance change has characteristics of a small channel and possesses a lifetime < or = 1 ms. To detect electric field-induced changes in the lipid-protein domains of PS II, we examined the effects of phospholipase A2 (PLA2) on EPL. Higher values of EPL were observed from vesicles that were exposed in the presence of PLA2 to an electroporating electric field than to a nonelectroporating electric field. The effect of the electroporating field was a long-lived one, lasting for a period > or = 2 min. This effect was attributed to long-lived electric field-induced structural changes in the lipid-protein domains of PS II. PMID:7811916

  15. Photosynthetic pigment localization and thylakoid membrane morphology are altered in Synechocystis 6803 phycobilisome mutants.

    PubMed

    Collins, Aaron M; Liberton, Michelle; Jones, Howland D T; Garcia, Omar F; Pakrasi, Himadri B; Timlin, Jerilyn A

    2012-04-01

    Cyanobacteria are oxygenic photosynthetic prokaryotes that are the progenitors of the chloroplasts of algae and plants. These organisms harvest light using large membrane-extrinsic phycobilisome antenna in addition to membrane-bound chlorophyll-containing proteins. Similar to eukaryotic photosynthetic organisms, cyanobacteria possess thylakoid membranes that house photosystem (PS) I and PSII, which drive the oxidation of water and the reduction of NADP+, respectively. While thylakoid morphology has been studied in some strains of cyanobacteria, the global distribution of PSI and PSII within the thylakoid membrane and the corresponding location of the light-harvesting phycobilisomes are not known in detail, and such information is required to understand the functioning of cyanobacterial photosynthesis on a larger scale. Here, we have addressed this question using a combination of electron microscopy and hyperspectral confocal fluorescence microscopy in wild-type Synechocystis species PCC 6803 and a series of mutants in which phycobilisomes are progressively truncated. We show that as the phycobilisome antenna is diminished, large-scale changes in thylakoid morphology are observed, accompanied by increased physical segregation of the two photosystems. Finally, we quantified the emission intensities originating from the two photosystems in vivo on a per cell basis to show that the PSI:PSII ratio is progressively decreased in the mutants. This results from both an increase in the amount of photosystem II and a decrease in the photosystem I concentration. We propose that these changes are an adaptive strategy that allows cells to balance the light absorption capabilities of photosystems I and II under light-limiting conditions.

  16. PSII-LHCII supercomplex organizations in photosynthetic membrane by coarse-grained simulation.

    PubMed

    Lee, Cheng-Kuang; Pao, Chun-Wei; Smit, Berend

    2015-03-12

    Green plant photosystem II (PSII) and light-harvesting complex II (LHCII) in the stacked grana regions of thylakoid membranes can self-organize into various PSII-LHCII supercomplexes with crystalline or fluid-like supramolecular structures to adjust themselves with external stimuli such as high/low light and temperatures, rendering tunable solar light absorption spectrum and photosynthesis efficiencies. However, the mechanisms controlling the PSII-LHCII supercomplex organizations remain elusive. In this work, we constructed a coarse-grained (CG) model of the thylakoid membrane including lipid molecules and a PSII-LHCII supercomplex considering association/dissociation of moderately bound-LHCIIs. The CG interaction between CG beads were constructed based on electron microscope (EM) experimental results, and we were able to simulate the PSII-LHCII supramolecular organization of a 500 × 500 nm(2) thylakoid membrane, which is compatible with experiments. Our CGMD simulations can successfully reproduce order structures of PSII-LHCII supercomplexes under various protein packing fractions, free-LHCII:PSII ratios, and temperatures, thereby providing insights into mechanisms leading to PSII-LHCII supercomplex organizations in photosynthetic membranes.

  17. Optical Signatures of Quantum Delocalization over Extended Domains in Photosynthetic Membranes.

    PubMed

    Schroeder, Christopher A; Caycedo-Soler, Felipe; Huelga, Susana F; Plenio, Martin B

    2015-08-27

    The prospect of coherent dynamics and excitonic delocalization across several light-harvesting structures in photosynthetic membranes is of considerable interest, but challenging to explore experimentally. Here we demonstrate theoretically that the excitonic delocalization across extended domains involving several light-harvesting complexes can lead to unambiguous signatures in the optical response, specifically, linear absorption spectra. We characterize, under experimentally established conditions of molecular assembly and protein-induced inhomogeneities, the optical absorption in these arrays from polarized and unpolarized excitation, and demonstrate that it can be used as a diagnostic tool to determine the resonance coupling between iso-energetic light-harvesting structures. The knowledge of these couplings would then provide further insight into the dynamical properties of transfer, such as facilitating the accurate determination of Förster rates.

  18. Gain and loss of photosynthetic membranes during plastid differentiation in the shoot apex of Arabidopsis.

    PubMed

    Charuvi, Dana; Kiss, Vladimir; Nevo, Reinat; Shimoni, Eyal; Adam, Zach; Reich, Ziv

    2012-03-01

    Chloroplasts of higher plants develop from proplastids, which are undifferentiated plastids that lack photosynthetic (thylakoid) membranes. In flowering plants, the proplastid-chloroplast transition takes place at the shoot apex, which consists of the shoot apical meristem (SAM) and the flanking leaf primordia. It has been believed that the SAM contains only proplastids and that these become chloroplasts only in the primordial leaves. Here, we show that plastids of the SAM are neither homogeneous nor necessarily null. Rather, their developmental state varies with the specific region and/or layer of the SAM in which they are found. Plastids throughout the L1 and L3 layers of the SAM possess fairly developed thylakoid networks. However, many of these plastids eventually lose their thylakoids during leaf maturation. By contrast, plastids at the central, stem cell-harboring region of the L2 layer of the SAM lack thylakoid membranes; these appear only at the periphery, near the leaf primordia. Thus, plastids in the SAM undergo distinct differentiation processes that, depending on their lineage and position, lead to either development or loss of thylakoid membranes. These processes continue along the course of leaf maturation.

  19. Photosynthetic control of electron transport and the regulation of gene expression.

    PubMed

    Foyer, Christine H; Neukermans, Jenny; Queval, Guillaume; Noctor, Graham; Harbinson, Jeremy

    2012-02-01

    The term 'photosynthetic control' describes the short- and long-term mechanisms that regulate reactions in the photosynthetic electron transport (PET) chain so that the rate of production of ATP and NADPH is coordinated with the rate of their utilization in metabolism. At low irradiances these mechanisms serve to optimize light use efficiency, while at high irradiances they operate to dissipate excess excitation energy as heat. Similarly, the production of ATP and NADPH in ratios tailored to meet demand is finely tuned by a sophisticated series of controls that prevents the accumulation of high NAD(P)H/NAD(P) ratios and ATP/ADP ratios that would lead to potentially harmful over-reduction and inactivation of PET chain components. In recent years, photosynthetic control has also been extrapolated to the regulation of gene expression because mechanisms that are identical or similar to those that serve to regulate electron flow through the PET chain also coordinate the regulated expression of genes encoding photosynthetic proteins. This requires coordinated gene expression in the chloroplasts, mitochondria, and nuclei, involving complex networks of forward and retrograde signalling pathways. Photosynthetic control operates to control photosynthetic gene expression in response to environmental and metabolic changes. Mining literature data on transcriptome profiles of C(3) and C(4) leaves from plants grown under high atmospheric carbon dioxide (CO(2)) levels compared with those grown with ambient CO(2) reveals that the transition to higher photorespiratory conditions in C(3) plants enhances the expression of genes associated with cyclic electron flow pathways in Arabidopsis thaliana, consistent with the higher ATP requirement (relative to NADPH) of photorespiration.

  20. Novel conducting polymer-heteropoly acid hybrid material for artificial photosynthetic membranes.

    PubMed

    McDonald, Michael B; Freund, Michael S

    2011-04-01

    Artificial photosynthetic (AP) approaches to convert and store solar energy will require membranes capable of conducting both ions and electrons while remaining relatively transparent and chemically stable. A new approach is applied herein involving previously described in situ chemical polymerization of electronically conducting poly(3,4-ethylenedioxythiophene) (PEDOT) in the presence of proton conducting heteropoly acid (HPA) phosphomolybdic acid (PMA). The electrochemical behaviour of the PEDOT/PMA hybrid material was investigated and it was found that the conducting polymer (CP) is susceptible to irreversible oxidative processes at potentials where water is oxidized. This will be problematic in AP devices should the process occur in very close proximity to a conducting polymer-based membrane. It was found that PEDOT grants the system good electrical performance in terms of conductivity and stability over a large pH window; however, the presence of PMA was not found to provide sufficient proton conductivity. This was addressed in an additional study by tuning the ionic (and in turn, electronic) conductivity in creating composites with the proton-permselective polymer Nafion. It was found that a material of this nature with near-equal conductivity for optimal chemical conversion efficiency will consist of roughly three parts Nafion and one part PEDOT/PMA.

  1. Oxygen and light effects on the expression of the photosynthetic apparatus in Bradyrhizobium sp. C7T1 strain.

    PubMed

    Montecchia, M S; Pucheu, N L; Kerber, N L; García, A F

    2006-12-01

    Photosynthetic bradyrhizobia are nitrogen-fixing symbionts colonizing the stem and roots of some leguminous plants like Aeschynomene. The effect of oxygen and light on the formation of the photosynthetic apparatus of Bradyrhizobium sp. C7T1 strain is described here. Oxygen is required for growth, but at high concentration inhibits the synthesis of bacteriochlorophyll (BChl) and of the photosynthetic apparatus. However, we show that in vitro, aerobic photosynthetic electron transport occurred leading to ADP photophosphorylation. The expression of the photosynthetic apparatus was regulated by oxygen in a manner which did not agree with earlier results in other photosynthetic bradyrhizobia since BChl accumulation was the highest under microaerobic conditions. This strain produces photosynthetic pigments when grown under cyclic illumination or darkness. However, under continuous white light illumination, a Northern blot analysis of the puf operon showed that, the expression of the photosynthetic genes of the antenna was considerable. Under latter conditions BChl accumulation in the cells was dependent on the oxygen concentration. It was not detectable at high oxygen tensions but became accumulated under low oxygen (microaerobiosis). It is known that in photosynthetic bradyrhizobia bacteriophytochrome photoreceptor (BphP) partially controls the synthesis of the photosystem in response to light. In C7T1 strain far-red light illumination did not stimulate the synthesis of the photosynthetic apparatus suggesting the presence of a non-functional BphP-mediated light regulatory mechanism.

  2. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  3. State of manganese in the photosynthetic apparatus. 2. X-ray absorption edge studies on manganese in photosynthetic membrane

    SciTech Connect

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

    1981-09-01

    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. In this study, 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.

  4. 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.

  5. Soluble variants of Rhodobacter capsulatus membrane-anchored cytochrome cy are efficient photosynthetic electron carriers.

    PubMed

    Oztürk, Yavuz; Lee, Dong-Woo; Mandaci, Sevnur; Osyczka, Artur; Prince, Roger C; Daldal, Fevzi

    2008-05-16

    Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt c(y). In species like R. capsulatus, cyt c(y) functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c(y) (S-c(y)), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt c(y). This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c(y) regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c(y) variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c(y) was about 340 mV, similar to that of cyts c2 or c(y). Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-c(y) was distinct from those seen with the cyt c2 or cyt c(y). The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c(y) interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-c(y) revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt c(y), whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.

  6. Eukaryotic behaviour of a prokaryotic energy-transducing membrane: fully detached vesicular organelles arise by budding from the Rhodobacter sphaeroides intracytoplasmic photosynthetic membrane.

    PubMed

    Niederman, Robert A

    2010-05-01

    A major feature that distinguishes prokaryotic organisms from eukaryotes is their less complex internal structure, in which all membrane-associated functions are thought to be present within a continuous lipid-protein bilayer, rather than with distinct organelles. Contrary to this notion, as described by Tucker and co-workers in this issue of Molecular Microbiology, the application of cryo-electron tomography to the purple bacterium Rhodobacter sphaeroides has demonstrated a heretofore unrecognized ultrastructural complexity within the intracytoplasmic membrane (ICM) housing the photosynthetic apparatus. In addition to distinguishing invaginations of the cytoplasmic membrane (CM) and interconnected vesicular structures still attached to the CM, a eukaryote-like ICM budding process was revealed, which results in the formation of fully detached vesicular structures. These bacterial organelles are able to carry out both the light-harvesting and light-driven energy transduction activities necessary for the cells to assume a photosynthetic lifestyle. Their formation is shown to represent the final stage in a membrane invagination and growth process, originating with small CM indentations, which after cell disruption give rise to a membrane fraction that can be separated from mature ICM vesicles by rate-zone sedimentation.

  7. Age-dependent changes in the functions and compositions of photosynthetic complexes in the thylakoid membranes of Arabidopsis thaliana.

    PubMed

    Nath, Krishna; Phee, Bong-Kwan; Jeong, Suyeong; Lee, Sun Yi; Tateno, Yoshio; Allakhverdiev, Suleyman I; Lee, Choon-Hwan; Nam, Hong Gil

    2013-11-01

    Photosynthetic complexes in the thylakoid membrane of plant leaves primarily function as energy-harvesting machinery during the growth period. However, leaves undergo developmental and functional transitions along aging and, at the senescence stage, these complexes become major sources for nutrients to be remobilized to other organs such as developing seeds. Here, we investigated age-dependent changes in the functions and compositions of photosynthetic complexes during natural leaf senescence in Arabidopsis thaliana. We found that Chl a/b ratios decreased during the natural leaf senescence along with decrease of the total chlorophyll content. The photosynthetic parameters measured by the chlorophyll fluorescence, photochemical efficiency (F v/F m) of photosystem II, non-photochemical quenching, and the electron transfer rate, showed a differential decline in the senescing part of the leaves. The CO2 assimilation rate and the activity of PSI activity measured from whole senescing leaves remained relatively intact until 28 days of leaf age but declined sharply thereafter. Examination of the behaviors of the individual components in the photosynthetic complex showed that the components on the whole are decreased, but again showed differential decline during leaf senescence. Notably, D1, a PSII reaction center protein, was almost not present but PsaA/B, a PSI reaction center protein is still remained at the senescence stage. Taken together, our results indicate that the compositions and structures of the photosynthetic complexes are differentially utilized at different stages of leaf, but the most dramatic change was observed at the senescence stage, possibly to comply with the physiological states of the senescence process.

  8. 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.

  9. cDNA cloning, expression levels and gene mapping of photosynthetic and non-photosynthetic ferredoxin genes in sunflower (Helianthus annuus L.).

    PubMed

    Venegas-Calerón, M; Zambelli, A; Ruiz-López, N; Youssar, L; León, A; Garcés, R; Martínez-Force, Enrique

    2009-03-01

    Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length ferredoxin cDNAs were cloned from sunflower (Helianthus annuus L.) leaves and developing seeds, HaFd1 and HaFd2, homologous to photosynthetic and non-photosynthetic ferredoxins, respectively. Based on these cDNAs, the respective genomic sequences were obtained and the presence of DNA polymorphisms was investigated. Complete sequencing of the HaFd1 and HaFd2 genes in different lines indicated the presence of two haplotypes for HaFd2 and their alignment showed that sequence polymorphisms are restricted to the 5'-NTR intron. In addition, specific DNA markers for the HaFd1 and HaFd2 genes were developed that enabled the genes to be mapped. Accordingly, the HaFd1 locus maps to linkage group 10 of the public sunflower map, while the HaFd2 locus maps to linkage group 11. Both ferredoxins display different spatial-temporal patterns of expression. While HaFd2 is expressed at similar levels in all tissues tested (leaves, stem, roots, cotyledons and developing seeds), HaFd1 is more strongly expressed in green tissues than in all the other tissues tested. Both photosynthetic- and heterotrophic-ferredoxins are present in sunflower seeds and may contribute to fatty acid desaturation during oil accumulation. Nevertheless, the levels of HaFd2 expression during seed formation are distinct in lines that only varied in the HaFd2 haplotypes they expressed.

  10. Light harvesting, energy transfer and electron cycling of a native photosynthetic membrane adsorbed onto a gold surface.

    PubMed

    Magis, Gerhard J; den Hollander, Mart-Jan; Onderwaater, Willem G; Olsen, John D; Hunter, C Neil; Aartsma, Thijs J; Frese, Raoul N

    2010-03-01

    Photosynthetic membranes comprise a network of light harvesting and reaction center pigment-protein complexes responsible for the primary photoconversion reactions: light absorption, energy transfer and electron cycling. The structural organization of membranes of the purple bacterial species Rb. sphaeroides has been elucidated in most detail by means of polarized light spectroscopy and atomic force microscopy. Here we report a functional characterization of native and untreated membranes of the same species adsorbed onto a gold surface. Employing fluorescence confocal spectroscopy and light-induced electrochemistry we show that adsorbed membranes maintain their energy and electron transferring functionality. Gold-adsorbed membranes are shown to generate a steady high photocurrent of 10 microA/cm(2) for several minutes and to maintain activity for up to three days while continuously illuminated. The surface-adsorbed membranes exhibit a remarkable functionality under aerobic conditions, even when exposed to light intensities well above that of direct solar irradiation. The component at the interface of light harvesting and electron cycling, the LH1 complex, displays exceptional stability, likely contributing to the robustness of the membranes. Peripheral light harvesting LH2 complexes show a light intensity dependent decoupling from photoconversion. LH2 can act as a reversible switch at low-light, an increased emitter at medium light and photobleaches at high light.

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

    PubMed

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

    1994-09-01

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

  12. 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.

  13. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  14. The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery.

    PubMed

    Grouneva, Irina; Rokka, Anne; Aro, Eva-Mari

    2011-12-02

    The thylakoid membrane of photoautotrophic organisms contains the main components of the photosynthetic electron transport chain. Detailed proteome maps of the thylakoid protein complexes of two marine diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, were created by means of two-dimensional blue native (BN)/SDS-PAGE coupled with mass spectrometry analysis. One novel diatom-specific photosystem I (PS I)-associated protein was identified. A second plastid-targeted protein with possible PS I interaction was discovered to be restricted to the centric diatom species T. pseudonana. PGR5/PGRL homologues were found to be the only protein components of PS I-mediated cyclic electron transport common to both species. For the first time, evidence for a possible PS I localization of LI818-like light harvesting proteins (Lhcx) is presented. This study also advances the current knowledge on the light harvesting antenna composition and Lhcx expression in T. pseudonana on the protein level and presents details on the molecular distribution of Lhcx in diatoms. Above mentioned proteins and several others with unknown function provide a broad basis for further mutagenesis analysis, aiming toward further understanding of the composition and function of the photosynthetic apparatus of diatoms. The proteomics approach of this study further served as a tool to confirm and improve genome-derived protein models.

  15. Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate.

    PubMed

    Manzo, Anthony J; Goushcha, Alexander O; Barabash, Yuri M; Kharkyanen, Valery N; Scott, Gary W

    2009-07-01

    Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I(exp), obeys a simple exponential law with the rate constant alphaI(exp) + k(rec), in which alpha is a parameter relating the light intensity, measured in mW/cm(2), to a corresponding theoretical rate in units of reciprocal seconds, and k(rec) is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the alpha parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer-Lambert-Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation.

  16. Atomic detail visualization of photosynthetic membranes with GPU-accelerated ray tracing

    SciTech Connect

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

    2015-12-12

    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. In this paper, 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. Finally, 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.

  17. 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

  18. Atomic detail visualization of photosynthetic membranes with GPU-accelerated ray tracing

    DOE PAGES

    Stone, John E.; Sener, Melih; Vandivort, Kirby L.; ...

    2015-12-12

    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. In this paper, 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. Finally, we describemore » 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.« less

  19. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  20. The effect of microgravity on proton permeability of thylakoid membranes and contribution of II and I photosystems in photosynthetic electron transport in pea chloroplasts.

    PubMed

    Zolotareva, E K; Onoiko, E B; Sytnik, S K; Podorvanov, V V

    1999-07-01

    According to a number investigations microgravity conditions affect membrane apparatus of photosynthesis in cells of higher plants and alga [for review, see Kordyum et al., 1994; Kordyum, 1997]. (see for review). Chloroplasts of space-grown pea plants showed disintegration of grana, shrinkage of the membrane constituting the grana stacks and other structural perturbance of the photosynthetic membranes. However there have been no studies on the effect of microgravity on proton permeability of thylakoid membranes and closely connected with this parameter their photochemical characteristics. The aim of the study is investigation of microgravity effects on protonic permeability of photosynthetic membrane and contribution of photosystem II (PSII) and photosystem I (PSI) in electron transfer from water to potassium ferrycianide (FeCy) in isolated pea chloroplasts. Pea.

  1. 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

  2. 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(-).

  3. 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

    2016-06-23

    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.

  4. Gain and Loss of Photosynthetic Membranes during Plastid Differentiation in the Shoot Apex of Arabidopsis[W

    PubMed Central

    Charuvi, Dana; Kiss, Vladimir; Nevo, Reinat; Shimoni, Eyal; Adam, Zach; Reich, Ziv

    2012-01-01

    Chloroplasts of higher plants develop from proplastids, which are undifferentiated plastids that lack photosynthetic (thylakoid) membranes. In flowering plants, the proplastid-chloroplast transition takes place at the shoot apex, which consists of the shoot apical meristem (SAM) and the flanking leaf primordia. It has been believed that the SAM contains only proplastids and that these become chloroplasts only in the primordial leaves. Here, we show that plastids of the SAM are neither homogeneous nor necessarily null. Rather, their developmental state varies with the specific region and/or layer of the SAM in which they are found. Plastids throughout the L1 and L3 layers of the SAM possess fairly developed thylakoid networks. However, many of these plastids eventually lose their thylakoids during leaf maturation. By contrast, plastids at the central, stem cell–harboring region of the L2 layer of the SAM lack thylakoid membranes; these appear only at the periphery, near the leaf primordia. Thus, plastids in the SAM undergo distinct differentiation processes that, depending on their lineage and position, lead to either development or loss of thylakoid membranes. These processes continue along the course of leaf maturation. PMID:22438022

  5. 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.

  6. 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

  7. Orientation and energy-transfer studies on chlorophyll in the photosynthetic membrane

    SciTech Connect

    Nairn, J.A.

    1981-12-01

    The two methods of study used for the light reactions of photosynthesis are orientation dependent spectroscopy and picosecond resolution of the fluorescence decay kinetics. Analysis of spectroscopic measurements on complex partially ordered ensembls, such as photosynthetic systems, is usually limited by knowledge of the orientational distribution function. A new method of parametrically representing the distribution function using a physical model of the partially ordered ensemble is described. The parametric representation of the distribution function is the density of states function. Many formulas are included which can be used to calculate density of state functions for a large range of problems. Fluorescence decay kinetics in chloroplasts from green plants and algae are investigated using a synchronously pumped, mode-locked dye laser as an excitation source.

  8. Demonstration of thermal dissipation of absorbed quanta during energy-dependent quenching of chlorophyll fluorescence in photosynthetic membranes.

    PubMed

    Yahyaoui, W; Harnois, J; Carpentier, R

    1998-11-27

    When plant leaves or chloroplasts are exposed to illumination that exceeds their photosynthetic capacity, photoprotective mechanisms such as described by the energy-dependent (non-photochemical) quenching of chlorophyll fluorescence are involved. The protective action is attributed to an increased rate constant for thermal dissipation of absorbed quanta. We applied photoacoustic spectroscopy to monitor thermal dissipation in spinach thylakoid membranes together with simultaneous measurement of chlorophyll fluorescence in the presence of inhibitors of opposite action on the formation of delta pH across the thylakoid membrane (tentoxin and nigericin/valinomycin). A linear relationship between the appearance of fluorescence quenching during formation of the delta pH and the reciprocal variation of thermal dissipation was demonstrated. Dicyclohexylcarbodiimide, which is known to prevent protonation of the minor light-harvesting complexes of photosystem II, significantly reduced the formation of fluorescence quenching and the concurrent increase in thermal dissipation. However, the addition of exogenous ascorbate to activate the xanthophyll de-epoxidase increased non-photochemical fluorescence quenching without affecting the measured thermal dissipation. It is concluded that a portion of energy-dependent fluorescence quenching that is independent of de-epoxidase activity can be readily measured by photoacoustic spectroscopy as an increase in thermal deactivation processes.

  9. 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

  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. Fluorescence imaging of single molecules and photosynthetic membranes with two-photon excitation

    SciTech Connect

    Sanchez, E.J.; Novotny, L.; Xie, X.S.

    1997-12-31

    We report the imaging of single-molecule fluorescence induced by two-photon excitation in ambient conditions. Using an inverted fluorescence microscope, we obtained the two-photon images of different single fluorophores (Rhodamine B, Sulforhodamine 101, Coumarin 535 on poly-methyl methacrylate films) and biological membrane fragments by Faster scanning the sample with respect to a diffraction limited focus of a mode-locked Ti: sapphire laser beam. The signal to background ratio was as high as 50:1 and the full width at half maximum (250nm) of a single-molecule peak was significantly shorter than that for one photon excitation. With its high sensitivity and simplicity, the two-photon experiment offers a valuable approach for spectroscopic studies on individual immobilized molecules.

  12. The isolation and characterization of a new iron-sulfur protein from photosynthetic membranes.

    PubMed

    Malkin, R; Aparicio, P J; Arnon, D I

    1974-06-01

    A new iron-sulfur protein, distinct from the soluble chloroplast ferredoxin, was isolated from chloroplast membranes. The isolated protein, purified to homogeneity, had a molecular weight of about 8000 and 4 atoms of iron and 4 inorganic sulfides per mole. Its absorption spectrum had a broad absorbance band in the 400 nm region, a shoulder at approximately 310 nm, and a peak around 280 nm. The absorbance ratio A(400) to A(280) was 0.55. The electron paramagnetic resonance spectrum (measured at 12 degrees K) of the reduced protein was similar to that of other reduced iron-sulfur proteins, showing a major resonance line at g = 1.94. The isolated protein, when photoreduced by spinach chloroplasts, can in turn transfer electrons to mammalian cytochrome c. However, the photoreduced protein cannot replace soluble ferredoxin in NADP(+) reduction because of its apparent inability to interact with the chloroplast enzyme, ferredoxin-NADP(+) reductase. The relation of the isolated iron-sulfur protein to the bound ferredoxin that acts as the primary electron acceptor in Photosystem I is discussed.

  13. The absence of chlorophyll b affects lateral mobility of photosynthetic complexes and lipids in grana membranes of Arabidopsis and barley chlorina mutants.

    PubMed

    Tyutereva, Elena V; Evkaikina, Anastasiia I; Ivanova, Alexandra N; Voitsekhovskaja, Olga V

    2017-04-05

    The lateral mobility of integral components of thylakoid membranes, such as plastoquinone, xanthophylls, and pigment-protein complexes, is critical for the maintenance of efficient light harvesting, high rates of linear electron transport, and successful repair of damaged photosystem II (PSII). The packaging of the photosynthetic pigment-protein complexes in the membrane depends on their size and stereometric parameters which in turn depend on the composition of the complexes. Chlorophyll b (Chlb) is an important regulator of antenna size and composition. In this study, the lateral mobility (the mobile fraction size) of pigment-protein complexes and lipids in grana membranes was analyzed in chlorina mutants of Arabidopsis and barley lacking Chlb. In the Arabidopsis ch1-3 mutant, diffusion of membrane lipids decreased as compared to wild-type plants, but the diffusion of photosynthetic complexes was not affected. In the barley chlorina f2 3613 mutant, the diffusion of pigment-protein complexes significantly decreased, while the diffusion of lipids increased, as compared to wild-type plants. We propose that the size of the mobile fractions of pigment-protein complexes in grana membranes in vivo is higher than reported previously. The data are discussed in the context of the protein composition of antennae, characteristics of the plastoquinone pool, and production of reactive oxygen species in leaves of chlorina mutants.

  14. Stability of integral membrane proteins under high hydrostatic pressure: the LH2 and LH3 antenna pigment-protein complexes from photosynthetic bacteria.

    PubMed

    Kangur, Liina; Timpmann, Kõu; Freiberg, Arvi

    2008-07-03

    The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure

  15. Advancing Rhodobacter sphaeroides as a platform for expression of functional membrane proteins.

    PubMed

    Erbakan, Mustafa; Curtis, Brandon S; Nixon, B Tracy; Kumar, Manish; Curtis, Wayne R

    2015-11-01

    Membrane protein overexpression is often hindered by toxic effects on the expression host, limiting achievable volumetric productivity. Moreover, protein structure and function may be impaired due to inclusion body formation and proteolytic degradation. To address these challenges, we employed the photosynthetic bacterium, Rhodobacter sphaeroides for expression of challenging membrane proteins including human aquaporin 9 (hAQP9), human tight junction protein occludin (Occ), Escherichia coli toxin peptide GhoT, cellulose synthase enzyme complex (BcsAB) of R. sphaeroides and cytochrome-cy (Cyt-cy) from Rhodobacter capsulatus. Titers of 47 mg/L for Cyt-cy, 7.5 mg/L for Occ, 1.5 mg/L for BcsAB and 0.5 mg/L for hAQP9 were achieved from affinity purification. While purification of GhoT was not successful, transformants displayed a distinct growth phenotype that correlated with GhoT expression. We also evaluated the functionality of these proteins by performing water transport studies for hAQP9, peroxidase activity for cytochrome-cy, and in vitro cellulose synthesis activity assay for BcsAB. While previous studies with Rhodobacter have utilized oxygen-limited semi-aerobic growth for membrane protein expression, substantial titer improvements are achieved as a result of a 3-fold increase in biomass yield using the anaerobic photoheterotrophic growth regime, which utilizes the strong native puc promoter. This versatile platform is shown to enable recovery of a wide variety of difficult-to-express membrane proteins in functional form.

  16. Analysis of Porphyra membrane transporters demonstrates gene transfer among photosynthetic eukaryotes and numerous sodium-coupled transport systems.

    PubMed

    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

    2012-04-01

    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.

  17. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  18. 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

  19. 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.

  20. 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.

  1. Enhanced cytokinin synthesis in tobacco plants expressing PSARK::IPT prevents the degradation of photosynthetic protein complexes during drought.

    PubMed

    Rivero, Rosa M; Gimeno, Jacinta; Van Deynze, Allen; Walia, Harkamal; Blumwald, Eduardo

    2010-11-01

    To identify genes associated with the cytokinin-induced enhanced drought tolerance, we analyzed the transcriptome of wild-type and transgenic tobacco (Nicotiana tabacum 'SR1') plants expressing P(SARK)::IPT (for senescence-associated receptor kinase::isopentenyltransferase) grown under well-watered and prolonged water deficit conditions using the tomato GeneChip. During water deficit, the expression of genes encoding components of the carotenoid pathway leading to ABA biosynthesis was enhanced in the wild-type plants, but repressed in the transgenic plants. On the other hand, transgenic plants displayed higher transcript abundance of genes involved in the brassinosteroid biosynthetic pathways. Several genes coding for proteins associated with Chl synthesis, light reactions, the Calvin-Benson cycle and photorespiration were induced in the transgenic plants. Notably, increased transcript abundance of genes associated with PSII, the cytochrome b(6)/f complex, PSI, NADH oxidoreductase and the ATP complex was found in the P(SARK)::IPT plants. The increased transcript abundance was assessed by quantitative PCR and the increased protein levels were confirmed by Western blots. Our results indicated that while the photosynthetic apparatus in the wild-type plants was degraded, photosynthesis in the transgenic plants was not affected and photosynthetic proteins were not degraded. During water deficit, wild-type plants displayed a significant reduction in electron transfer and photochemical quenching, with a marked increase in non-photochemical quenching, suggesting a decrease in energy transfer to the PSII core complexes and an increase in cyclic electron transfer reactions.

  2. Expression of basement membrane antigens in spindle cell melanoma.

    PubMed

    Prieto, V G; Woodruff, J M

    1998-07-01

    Spindle cell melanoma (SCM) is an uncommon form of melanoma that may be confused histologically with other tumors, including malignant peripheral nerve sheath tumors (MPNST). Tumors with neural differentiation and melanocytic nevi may both show basement membrane immunohistochemically and at the ultrastructural level. However, most ultrastructural studies of melanoma have failed to demonstrate well formed basement membrane around tumor cells. The presence of basement membrane has been used by some authors as evidence favoring MPNST, as opposed to SCM. To evaluate this distinction immunohistochemically, 22 primary and metastatic cutaneous melanomas having a spindle cell component (SCM) were studied using monoclonal antibodies against laminin and Type IV collagen. S100 protein and HMB45 antigen expression were also studied. All but one of the SCM were reactive for S100 protein in at least 25% of the cells. Thirteen of 20 tumors (65%) were focally reactive with HMB45. Laminin was expressed in 42% of the tumors (only membranous pattern in 3; cytoplasmic and membranous in 5). Seventeen tumors (77%) expressed type IV collagen (only membranous pattern in 7; cytoplasmic and membranous pattern in 10). Laminin and type IV collagen, known components of basement membrane, are often found in SCM. Therefore, their detection cannot be used to distinguish SCM from MPNST.

  3. Thrombospondin expression in myofibers stabilizes muscle membranes

    PubMed Central

    Vanhoutte, Davy; Schips, Tobias G; Kwong, Jennifer Q; Davis, Jennifer; Tjondrokoesoemo, Andoria; Brody, Matthew J; Sargent, Michelle A; Kanisicak, Onur; Yi, Hong; Gao, Quan Q; Rabinowitz, Joseph E; Volk, Talila; McNally, Elizabeth M; Molkentin, Jeffery D

    2016-01-01

    Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of βPS integrin. This functional conservation emphasizes the fundamental importance of Thbs’ as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy. DOI: http://dx.doi.org/10.7554/eLife.17589.001 PMID:27669143

  4. Constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase enhances stomatal opening and photosynthetic performance of Arabidopsis thaliana.

    PubMed

    Kebeish, Rashad; Niessen, Markus; Oksaksin, Mehtap; Blume, Christian; Peterhaensel, Christoph

    2012-02-01

    The effect of constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase (PEPC) on the opening state of stomata and photosynthetic performance in Arabidopsis thaliana plants was studied. Transcript accumulation analyses of the A. thaliana dark-induced (Din10 and Din6) and the Pisum sativum asparagine synthetase 2 promoters (Asn2) in transiently transformed tobacco leaves showed that Din10 promoter induced more DsRed accumulation in the dark compared to the other din genes. Overexpression of PEPC under the control of the constitutive enhanced CaMV 35S (p35SS) and dark-induced Din10 promoter in stably transformed A. thaliana plants increased the number of opened stomata in dark adapted leaves. Gas exchange measurements using A. thaliana plants transgenic for p35SS-PEPC and Din10-PEPC revealed a marked increase in stomatal conductance, transpiration, and dark respiration rates measured in the dark compared to wild-type plants. Moreover, measurement of CO(2) assimilation rates at different external CO(2) concentrations (C(a) ) and different light intensities shows an increase in the CO(2) assimilation rates in transgenic Arabidopsis lines compared to wild-type plants. This is considered as first step towards transferring the aspects of Crassulacean acid metabolism-like photosynthetic mechanism into C3 plants.

  5. Influence of heat stress on leaf ultrastructure, photosynthetic performance, and ascorbate peroxidase gene expression of two pear cultivars (Pyrus pyrifolia).

    PubMed

    Liu, Dong-feng; Zhang, Dong; Liu, Guo-qin; Hussain, Sayed; Teng, Yuan-wen

    2013-12-01

    Plants encounter a variety of stresses in natural environments. One-year-old pot-grown trees of pear (Pyrus pyrifolia Nakai cv. Cuiguan and Wonhwang) were exposed to two heat stress regimes. Under constant short-term heat stress, chloroplasts and mitochondria were visibly damaged. Relative chlorophyll content and maximum photochemical efficiency of photosystem II were significantly decreased, which indicated that the leaf photosynthetic capability declined. Under chronic heat stress, mesophyll cell ultrastructure was not obviously damaged, but leaf photosynthetic capability was still restrained. As chronic heat stress was a simulation of the natural environment in summer, further study of the responses under this stress regime was undertaken. Ascorbate peroxidase (APX) activity was increased in 'Cuiguan', but not in 'Wonhwang'. Inducible expression of PpAPX genes in the cytoplasm, chloroplasts and peroxisomes was consistent with increased APX activity in 'Cuiguan', whereas only weak induction of PpAPX genes was observed in 'Wonhwang'. The isoenzymes cytosolic APX1 (cAPX1) and stromal APX (sAPX) were confirmed to be localized in the cytoplasm and chloroplasts, respectively.

  6. Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane-anchored EGFP.

    PubMed

    Deng, Yaqi; Kim, BongWoo; He, Xuelian; Kim, Sunja; Lu, Changqing; Wang, Haibo; Cho, Ssang-Goo; Hou, Yiping; Li, Jianrong; Zhao, Xianghui; Lu, Q Richard

    2014-04-01

    Myelinogenesis is a complex process that involves substantial and dynamic changes in plasma membrane architecture and myelin interaction with axons. Highly ramified processes of oligodendrocytes in the central nervous system (CNS) make axonal contact and then extrapolate to wrap around axons and form multilayer compact myelin sheathes. Currently, the mechanisms governing myelin sheath assembly and axon selection by myelinating cells are not fully understood. Here, we generated a transgenic mouse line expressing the membrane-anchored green fluorescent protein (mEGFP) in myelinating cells, which allow live imaging of details of myelinogenesis and cellular behaviors in the nervous systems. mEGFP expression is driven by the promoter of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNP) that is expressed in the myelinating cell lineage. Robust mEGFP signals appear in the membrane processes of oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS), wherein mEGFP expression defines the inner layers of myelin sheaths and Schmidt-Lanterman incisures in adult sciatic nerves. In addition, mEGFP expression can be used to track the extent of remyelination after demyelinating injury in a toxin-induced demyelination animal model. Taken together, the membrane-anchored mEGFP expression in the new transgenic line would facilitate direct visualization of dynamic myelin membrane formation and assembly during development and process remodeling during remyelination after various demyelinating injuries.

  7. Light-harvesting mutants show differential gene expression upon shift to high light as a consequence of photosynthetic redox and reactive oxygen species metabolism.

    PubMed

    Tikkanen, Mikko; Gollan, Peter J; Mekala, Nageswara Rao; Isojärvi, Janne; Aro, Eva-Mari

    2014-04-19

    The amount of light energy that is harvested and directed to the photosynthetic machinery is regulated in order to control the production of reactive oxygen species (ROS) in leaf tissues. ROS have important roles as signalling factors that instigate and mediate a range of cellular responses, suggesting that the mechanisms regulating light-harvesting and photosynthetic energy transduction also affect cell signalling. In this study, we exposed wild-type (WT) Arabidopsis and mutants impaired in the regulation of photosynthetic light-harvesting (stn7, tap38 and npq4) to transient high light (HL) stress in order to study the role of these mechanisms for up- and downregulation of gene expression under HL stress. The mutants, all of which have disturbed regulation of excitation energy transfer and distribution, responded to transient HL treatment with surprising similarity to the WT in terms of general 'abiotic stress-regulated' genes associated with hydrogen peroxide and 12-oxo-phytodienoic acid signalling. However, we identified distinct expression profiles in each genotype with respect to induction of singlet oxygen and jasmonic acid-dependent responses. The results of this study suggest that the control of excitation energy transfer interacts with hormonal regulation. Furthermore, the photosynthetic pigment-protein complexes appear to operate as receptors that sense the energetic balance between the photosynthetic light reactions and downstream metabolism.

  8. The Lack of Lutein Accelerates the Extent of Light-induced Bleaching of Photosynthetic Pigments in Thylakoid Membranes of Arabidopsis thaliana.

    PubMed

    Dobrev, Konstantin; Stanoeva, Daniela; Velitchkova, Maya; Popova, Antoaneta V

    2016-05-01

    The high light-induced bleaching of photosynthetic pigments and the degradation of proteins of light-harvesting complexes of PSI and PSII were investigated in isolated thylakoid membranes of Arabidopsis thaliana, wt and lutein-deficient mutant lut2, with the aim of unraveling the role of lutein for the degree of bleaching and degradation. By the means of absorption spectroscopy and western blot analysis, we show that the lack of lutein leads to a higher extent of pigment photobleaching and protein degradation in mutant thylakoid membranes in comparison with wt. The highest extent of bleaching is suffered by chlorophyll a and carotenoids, while chlorophyll b is bleached in lut2 thylakoids during long periods at high illumination. The high light-induced degradation of Lhca1, Lhcb2 proteins and PsbS was followed and it is shown that Lhca1 is more damaged than Lhcb2. The degradation of analyzed proteins is more pronounced in lut2 mutant thylakoid membranes. The lack of lutein influences the high light-induced alterations in organization of pigment-protein complexes as revealed by 77 K fluorescence.

  9. Photosynthetic plasticity in Flaveria brownii: Growth irradiance and the expression of C sub 4 photosynthesis

    SciTech Connect

    Cheng, Shuhua; Moore, B.D.; Wu, Jingrui; Edwards, G.E.; Ku, M.S.B. )

    1989-04-01

    Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO{sub 2} compensation point and the inhibition of photosynthesis by 21% O{sub 2} were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C{sub 4} cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO{sub 2} compensation point and the degree of O{sub 2} inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO{sub 2} between Rubisco of the C{sub 3} pathway and phosphoenolpyruvate carboxylase of the C{sub 4} cycle was determined by exposing leaves to {sup 14}CO{sub 2} for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that {approximately}94% of the CO{sub 2} was fixed by the C{sub 4} cycle in high light grown plants, versus {approximately}78% in low light grown plants. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C{sub 4}-like pattern of compartmentation. Pyruvate,Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants.

  10. 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

  11. [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.

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

    PubMed

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

    2014-11-01

    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

  13. Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (Oryza sativa).

    PubMed

    Chang, Hsiang; Huang, Hsiang-En; Cheng, Chin-Fu; Ho, Mei-Hsuan; Ger, Mang-Jye

    2017-04-01

    The plant ferredoxin-like protein (PFLP) gene, cloned from sweet peppers predicted as an electron carrier in photosynthesis, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Most of pflp related studies focused on anti-pathogenic effects, while less understanding for the effects in photosynthesis with physiological aspects, such as photosynthesis rate, and levels of carbohydrate metabolites. This project focuses on the effects of pflp overexpression on photosynthesis by physiological evaluations of carbon assimilation with significant higher levels of carbohydrates with higher photosynthesis efficiency. In this report, two independent transgenic lines of rice plants (designated as pflp-1 and pflp-2) were generated from non-transgenic TNG67 rice plant (WT). Both transgenic pflp rice plants exhibited enhanced photosynthesis efficiency, and gas exchange rates of photosynthesis were 1.3- and 1.2-fold higher for pflp-1 and pflp-2 than WT respectively. Significantly higher electron transport rates of pflp rice plants were observed. Moreover, photosynthetic products, such as fructose, glucose, sucrose and starch contents of pflp transgenic lines were increased accordingly. Molecular evidences of carbohydrate metabolism related genes activities (osHXK5, osHXK6, osAGPL3, osAGPS2α, osSPS, ospFBPase, oscFBPase, and osSBPase) in transgenic lines were higher than those of WT. For performance of crop production, 1000-grain weight for pflp-1 and pflp-2 rice plants were 52.9 and 41.1 g that were both significantly higher than 31.6 g for WT, and panicles weights were 1.4- and 1.2-fold higher than WT. Panicle number, tiller number per plants for pflp rice plants were all significantly higher compared with those of WT where there was no significant difference observed between two pflp rice plants. Taken altogether; this study demonstrated that constitutive pflp expression can improve rice production by

  14. Differential regulation of Arabidopsis plastid gene expression and RNA editing in non-photosynthetic tissues.

    PubMed

    Tseng, Ching-Chih; Lee, Chih-Jen; Chung, Yi-Ting; Sung, Tzu-Ying; Hsieh, Ming-Hsiun

    2013-07-01

    RNA editing is one of the post-transcriptional processes that commonly occur in plant plastids and mitochondria. In Arabidopsis, 34 C-to-U RNA editing events, affecting transcripts of 18 plastid genes, have been identified. Here, we examined the editing and expression of these transcripts in different organs, and in green and non-green seedlings (etiolated, cia5-2, ispF and ispG albino mutants, lincomycin-, and norflurazon-treated). The editing efficiency of Arabidopsis plastid transcripts varies from site to site, and may be specifically regulated in different tissues. Steady state levels of plastid transcripts are low or undetectable in etiolated seedlings, but most editing sites are edited with efficiencies similar to those observed in green seedlings. By contrast, the editing of some sites is completely lost or significantly reduced in other non-green tissues; for instance, the editing of ndhB-149, ndhB-1255, and ndhD-2 is completely lost in roots and in lincomycin-treated seedlings. The editing of ndhD-2 is also completely lost in albino mutants and norflurazon-treated seedlings. However, matK-640 is completely edited, and accD-794, atpF-92, psbE-214, psbF-77, psbZ-50, and rps14-50 are completely or highly edited in both green and non-green tissues. In addition, the expression of nucleus-encoded RNA polymerase dependent transcripts is specifically induced by lincomycin, and the splicing of ndhB transcripts is significantly reduced in the albino mutants and inhibitor-treated seedlings. Our results indicate that plastid gene expression, and the splicing and editing of plastid transcripts are specifically and differentially regulated in various types of non-green tissues.

  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. Epithelial membrane protein 1 expression in ovarian serous tumors.

    PubMed

    Demirag, Guzin Gonullu; Kefeli, Mehmet; Kemal, Yasemin; Yucel, Idris

    2016-03-01

    The present study aimed to analyze the clinical significance of epithelial membrane protein 1 (EMP1) expression in ovarian serous tumors. A total of 84 cases of ovarian serous tumor (50 patients with malignant ovarian serous tumors and 34 patients with borderline and benign serous tumors) were retrospectively analyzed. Differences in the expression levels of EMP1 between the malignant and non-malignant tumor groups were evaluated by immunohistochemical staining. In addition, the association between EMP1 expression and prognostic factors in malignant ovarian serous tumors was investigated. The expression levels of EMP1 were significantly reduced in all the 50 malignant ovarian serous tumors, compared with the 34 non-malignant ovarian serous tumors (P<0.000). Reduced expression of EMP1 was correlated with high grade (P=0.009) and stage (P<0.000) of malignant tumors. EMP1 expression was not observed to be correlated with any other investigated parameters, including surgery, type of operation and chemotherapy response (P>0.005). These results indicated that EMP1 may have a significant role as a negative regulator in ovarian serous tumors, and reduced EMP1 expression in serous tumors may be associated with increased disease severity.

  17. Directed assembly of defined oligomeric photosynthetic reaction centres through adaptation with programmable extra-membrane coiled-coil interfaces.

    PubMed

    Swainsbury, David J K; Harniman, Robert L; Di Bartolo, Natalie D; Liu, Juntai; Harper, William F M; Corrie, Alexander S; Jones, Michael R

    2016-12-01

    A challenge associated with the utilisation of bioenergetic proteins in new, synthetic energy transducing systems is achieving efficient and predictable self-assembly of individual components, both natural and man-made, into a functioning macromolecular system. Despite progress with water-soluble proteins, the challenge of programming self-assembly of integral membrane proteins into non-native macromolecular architectures remains largely unexplored. In this work it is shown that the assembly of dimers, trimers or tetramers of the naturally monomeric purple bacterial reaction centre can be directed by augmentation with an α-helical peptide that self-associates into extra-membrane coiled-coil bundle. Despite this induced oligomerisation the assembled reaction centres displayed normal spectroscopic properties, implying preserved structural and functional integrity. Mixing of two reaction centres modified with mutually complementary α-helical peptides enabled the assembly of heterodimers in vitro, pointing to a generic strategy for assembling hetero-oligomeric complexes from diverse modified or synthetic components. Addition of two coiled-coil peptides per reaction centre monomer was also tolerated despite the challenge presented to the pigment-protein assembly machinery of introducing multiple self-associating sequences. These findings point to a generalised approach where oligomers or longer range assemblies of multiple light harvesting and/or redox proteins can be constructed in a manner that can be genetically-encoded, enabling the construction of new, designed bioenergetic systems in vivo or in vitro.

  18. 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.

  19. 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.

  20. Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy.

    PubMed

    Rood, Ilse M; Merchant, Michael L; Wilkey, Daniel W; Zhang, Terry; Zabrouskov, Vlad; van der Vlag, Johan; Dijkman, Henry B; Willemsen, Brigith K; Wetzels, Jack F; Klein, Jon B; Deegens, Jeroen K

    2015-11-01

    Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein-2 (LIMP-2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP-2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co-localization of LIMP-2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP-2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP-2 in glomeruli of patients with iMN.

  1. Effect of light and oxygen and adaptation to changing light conditions in a photosynthetic mutant in which the LHII complex of Rhv. sulfidophilum was heterologously expressed in a strain of Rb. capsulatus whose puc operon was deleted.

    PubMed

    Barbieri Md, María del Rosario; Kerber, Norma L; Pucheu, Norma L; Tadros, Monier H; García, Augusto F

    2002-09-01

    In this paper we show the effect of oxygen and light on the expression of the photosynthetic apparatus of a mutant heterologously expressing the puc operon. This mutant was obtained by introducing in trans an expression plasmid, bearing the puc A, B, and C genes of Rhv. sulfidophilum, as well as its own promoter, in an LHII(-) mutant of Rb. capsulatus. The results showed that oxygen and light repressed LHII expression. Even low-light intensities lowered the LHII content to undetectable levels by spectrophotometry or by SDS-PAGE. In high-light grown cells, where the relative ratios of LHI and LHII complexes were significantly diminished, we were able to detect LHII complexes. Under the latter condition, the absorption spectrum showed that some pigment accumulated in the membrane even in the absence of cell division. These pigments were used in a later step to assemble LHII complexes, when the high-light grown cells were transferred to semiaerobiosis in the dark. Transition of high-light grown cells to low-light conditions allowed us to study the adaptability of these heterologous mutant cells. We observed that adaptation never occurred, in part probably owing to energy limitation.

  2. Hydrogen sulphide enhances photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings.

    PubMed

    Chen, Juan; Wu, Fei-Hua; Wang, Wen-Hua; Zheng, Chen-Juan; Lin, Guang-Hui; Dong, Xue-Jun; He, Jun-Xian; Pei, Zhen-Ming; Zheng, Hai-Lei

    2011-08-01

    Hydrogen sulphide (H(2)S) is emerging as a potential messenger molecule involved in modulation of physiological processes in animals and plants. In this report, the role of H(2)S in modulating photosynthesis of Spinacia oleracea seedlings was investigated. The main results are as follows. (i) NaHS, a donor of H(2)S, was found to increase the chlorophyll content in leaves. (ii) Seedlings treated with different concentrations of NaHS for 30 d exhibited a significant increase in seedling growth, soluble protein content, and photosynthesis in a dose-dependent manner, with 100 μM NaHS being the optimal concentration. (iii) The number of grana lamellae stacking into the functional chloroplasts was also markedly increased by treatment with the optimal NaHS concentration. (iv) The light saturation point (Lsp), maximum net photosynthetic rate (Pmax), carboxylation efficiency (CE), and maximal photochemical efficiency of photosystem II (F(v)/F(m)) reached their maximal values, whereas the light compensation point (Lcp) and dark respiration (Rd) decreased significantly under the optimal NaHS concentration. (v) The activity of ribulose-1,5-bisphosphate carboxylase (RuBISCO) and the protein expression of the RuBISCO large subunit (RuBISCO LSU) were also significantly enhanced by NaHS. (vi) The total thiol content, glutathione and cysteine levels, internal concentration of H(2)S, and O-acetylserine(thiol)lyase and L-cysteine desulphydrase activities were increased to some extent, suggesting that NaHS also induced the activity of thiol redox modification. (vii) Further studies using quantitative real-time PCR showed that the gene encoding the RuBISCO large subunit (RBCL), small subunit (RBCS), ferredoxin thioredoxin reductase (FTR), ferredoxin (FRX), thioredoxin m (TRX-m), thioredoxin f (TRX-f), NADP-malate dehydrogenase (NADP-MDH), and O-acetylserine(thiol)lyase (OAS) were up-regulated, but genes encoding serine acetyltransferase (SERAT), glycolate oxidase (GYX), and cytochrome

  3. 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.

  4. Heterologous expression and purification of membrane-bound pyrophosphatases.

    PubMed

    Kellosalo, J; Kajander, T; Palmgren, M G; Lopéz-Marqués, R L; Goldman, A

    2011-09-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low light intensity. While they are completely absent in mammalians, they are key players in the survival of disease-causing protozoans making these proteins attractive pharmacological targets. In this work, we aimed at the purification of M-PPases in amounts suitable for crystallization as a first step to obtain structural information for drug design. We have tested the expression of eight integral membrane pyrophosphatases in Saccharomyces cerevisiae, six from bacterial and archaeal sources and two from protozoa. Two proteins originating from hyperthermophilic organisms were purified in dimeric and monodisperse active states. To generate M-PPases with an increased hydrophilic surface area, which potentially should facilitate formation of crystal contacts, phage T4 lysozyme was inserted into different extramembraneous loops of one of these M-PPases. Two of these fusion proteins were active and expressed at levels that would allow their purification for crystallization purposes.

  5. 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.

  6. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

  7. 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.

  8. Expression of the Minor Isoform Pea Ferredoxin in Tobacco Alters Photosynthetic Electron Partitioning and Enhances Cyclic Electron Flow1[W

    PubMed Central

    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.

    2013-01-01

    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

  9. Vaccinia virus virion membrane biogenesis protein A11 associates with viral membranes in a manner that requires the expression of another membrane biogenesis protein, A6.

    PubMed

    Wu, Xiang; Meng, Xiangzhi; Yan, Bo; Rose, Lloyd; Deng, Junpeng; Xiang, Yan

    2012-10-01

    A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.

  10. New examples of membrane protein expression and purification using the yeast based Pdr1-3 expression strategy.

    PubMed

    Gupta, Rakeshkumar P; Kueppers, Petra; Schmitt, Lutz

    2014-12-10

    Overexpression and purification of membrane proteins has been a bottleneck for their functional and structural study for a long time. Both homologous and heterologous expression of membrane proteins with suitable tags for purification presents unique challenges for cloning and expression. Saccharomyces cerevisiae is a potential host system with significant closeness to higher eukaryotes and provides opportunity for attempts to express membrane proteins. In the past, bakers yeast containing mutations within the transcriptional regulator Pdr1 has been used to overexpress various membrane proteins including for example the ABC transporters Pdr5 and Yor1, respectively. In this study we exploited this system and tried to express and purify 3 membrane proteins in yeast along with Pdr5 and Yor1 viz. Rsb1, Mdl1 and Drs2 by virtue of an N-terminal 14-histidine affinity tag. Out of these five, we could express all membrane proteins although at different levels. Satisfactory yields were obtained for three examples i.e. Pdr5, Yor1 and Drs2. Rsb1 expression was comparatively low and Mdl1 was rather unsatisfactory. Thus, we demonstrate here the application of this yeast based expression system that is suitable for cloning, expression and purification of a wide variety of membrane proteins.

  11. [The heterologous expression and purification of membrane protein from Mycobacterium tuberculosis].

    PubMed

    Liao, Dan; Xie, Jian-Ping; Wang, Hong-Hai

    2007-10-01

    Membrane proteins fulfill a wide range of central functions in the cell, but their structure determination remains one of the great challenges in structural biology. The heterologous overexpression is a demanding task. Here, we provide an overview of recent advance to heterologous expression and purification of membrane protein from Mycobacterium tuberculosis, whose membrane proteins represent the majority of the new potential drug targets in this bacillus, which is ranked as the number1 cause of infectious disease mortality in the world. A detailed structural and functional understanding of the membranes protein of Mycobacterium tuberculosis will be critical both for an understanding of the biology of infection and for the rational development of novel therapeutics. The procedures for functional expression followed by purification of membranes protein are reviewed here together with nonfunctional expression in inclusion bodies and subsequent refolding to produce functional proteins. The new expression systems, new approaches to soluble expression of recombinant proteins, new methods for membrane protein folding in vitro and new purification technology will provide a basis for choosing the best expression and purification protocol for a given membrane protein. The goal of this review is to aid researchers in the choice of a suitable expression system for their favourite proteins and make overproduction of functional membrane proteins becomes easier.

  12. Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

    PubMed

    Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

    2014-01-22

    Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes.

  13. Lipids in photosynthetic reaction centres: structural roles and functional holes.

    PubMed

    Jones, Michael R

    2007-01-01

    Photosynthetic proteins power the biosphere. Reaction centres, light harvesting antenna proteins and cytochrome b(6)f (or bc(1)) complexes are expressed at high levels, have been subjected to an intensive spectroscopic, biochemical and mutagenic analysis, and several have been characterised to an informatively high resolution by X-ray crystallography. In addition to revealing the structural basis for the transduction of light energy, X-ray crystallography has brought molecular insights into the relationships between these multicomponent membrane proteins and their lipid environment. Lipids resolved in the X-ray crystal structures of photosynthetic proteins bind light harvesting cofactors, fill intra-protein cavities through which quinones can diffuse, form an important part of the monomer-monomer interface in multimeric structures and may facilitate structural flexibility in complexes that undergo partial disassembly and repair. It has been proposed that individual lipids influence the biophysical properties of reaction centre cofactors, and so affect the rate of electron transfer through the complex. Lipids have also been shown to be important for successful crystallisation of photosynthetic proteins. Comparison of the three types of reaction centre that have been structurally characterised reveals interesting similarities in the position of bound lipids that may point towards a generic requirement to reinforce the structure of the core electron transfer domain. The crystallographic data are also providing new opportunities to find molecular explanations for observed effects of different types of lipid on the structure, mechanism and organisation of reaction centres and other photosynthetic proteins.

  14. Progesterone receptor membrane component 1 (PGRMC1) expression in fetal membranes among women with preterm premature rupture of the membranes (PPROM).

    PubMed

    Feng, L; Antczak, B C; Lan, L; Grotegut, C A; Thompson, J L; Allen, T K; Murtha, A P

    2014-05-01

    PGRMC1 function is implicated in maintaining fetal membrane (FM) integrity. PGRMC1 was detectable primarily in the cytoplasm of FM cells and was actively regulated in FMs and relevant for PGRMC1-mediated progesterone action. By cell type, PGRMC1 expression was higher in amnion and chorion compared with decidua. By clinical phenotype, PGRMC1 expression was higher among preterm-no-labor and term-no-labor subjects compared to PPROM. PGRMC1 expression appears to be diminished in PPROM subjects.

  15. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    PubMed Central

    Findlay, Heather E; McClafferty, Heather; Ashley, Richard H

    2005-01-01

    Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide

  16. Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil.

    PubMed

    Ivanova, L A; Ronzhina, D A; Ivanov, L A; Stroukova, L V; Peuke, A D; Rennenberg, H

    2011-07-01

    Recent studies of transgenic poplars over-expressing the genes gsh1 and gsh2 encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO(2) diffusion, chlorophyll and carbohydrate content in wild-type poplar and transgenic plants over-expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal-contaminated soil in the field. Over-expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6-fold leaf area per leaf compared to wild-type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over-expression of γ-ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3-fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild-type plants but not in transformants. Biomass accumulation of wild-type poplars decreased in contaminated soil by more than 30-fold, whereas transformants showed a twofold decrease

  17. CO2 reduction and organic compounds production by photosynthetic bacteria with surface displayed carbonic anhydrase and inducible expression of phosphoenolpyruvate carboxylase.

    PubMed

    Park, Ju-Yong; Kim, Yang-Hoon; Min, Jiho

    2017-01-01

    In Rhodobacter sphaeroides, carbonic anhydrase (CA; EC 4.2.1.1) is a zinc-containing metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-) while phosphoenolpyruvate carboxylase (PEPC; 4.1.1.31), an enzyme involved in the carbon metabolism that catalyzed the fixation of CO2 to PEP, is a key factor for biological fixation of CO2 and enhances the production of organic compounds. In this study, the recombinant R. sphaeroides with highly-expressed CA was developed based on a surface displayed system of CA (pJY-OmpCA) on the outer membrane of R. sphaeroides using outer membrane protein (Omp) in R. sphaeroides, Finally, two more different recombinant R. sphaeroides were developed, which transformed with a two-vector system harboring cytosolic expressed CA (pJY-OmpCA-CA)or PEPC (pJY-OMPCA-PEPC) in R. sphaeroides with surface displayed CA on the outer membrane. In case of recombinant R. sphaeroides with the pJY-OmpCA-PEPC, it has shown the highest CO2 reduction efficiency and the production of several organic compounds (carotenoids, polyhydroxybutyrate, malic acid, succinic acid). It means that the surface displayed CA on the R. sphaeroides would accelerate the CO2-bicabonate conversion on the bacterial outer membrane. Moreover, inducible over-expression of PEPC with surface-displayed CA was successfully used to facilitate a rapider CO2 reduction and quicker production of organic compounds.

  18. 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.

  19. Functional expression of mammalian receptors and membrane channels in different cells.

    PubMed

    Eifler, Nora; Duckely, Myriam; Sumanovski, Lazar T; Egan, Terrance M; Oksche, Alexander; Konopka, James B; Lüthi, Anita; Engel, Andreas; Werten, Paul J L

    2007-08-01

    In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.

  20. Rational design of a fusion partner for membrane protein expression in E. coli

    PubMed Central

    Luo, Jianying; Choulet, Julie; Samuelson, James C

    2009-01-01

    We have designed a novel protein fusion partner (P8CBD) to utilize the co-translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP-dependence was demonstrated by analyzing the membrane translocation of P8CBD-PhoA fusion proteins in wt and SRP-ffh77 mutant cells. We also demonstrate that the P8CBD N-terminal fusion partner promotes over-expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over-expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane-associated protein. PMID:19530231

  1. Expression of mammalian membrane proteins in mammalian cells using Semliki Forest virus vectors.

    PubMed

    Lundstrom, Kenneth

    2010-01-01

    One of the major bottlenecks in drug screening and structural biology on membrane proteins has for a long time been the expression of recombinant protein in sufficient quality and quantity. The expression has been evaluated in all existing expression systems, from cell-free translation and bacterial systems to expression in animal cells. In contrast to soluble proteins, the expression levels have been relatively low due to the following reasons: The topology of membrane proteins requires special, posttranslational processing, folding, and insertion into membranes, which often are mammalian cell specific. Despite these strict demands, functional membrane proteins (G protein-coupled receptors, ion channels, and transporters) have been successfully expressed in bacterial, yeast, and insect cells. A general drawback observed in prokaryotic cells is that accumulation of foreign protein in membranes is toxic and results in growth arrest and therefore low yields of recombinant protein.In this chapter, the focus is on expression of recombinant mammalian membrane proteins in mammalian host cells, particularly applying Semliki Forest virus (SFV) vectors. Replication-deficient SFV vectors are rapidly generated at high titers in BHK-21 (Baby Hamster Kidney) cells, which then are applied for a broad range of mammalian and nonmammalian cells. The SFV system has provided high expression levels of topologically different proteins, especially for membrane proteins. Robust ligand-binding assays and functional coupling to G proteins and electrophysiological recordings have made the SFV system an attractive tool in drug discovery. Furthermore, the high susceptibility of SFV vectors to primary neurons has allowed various applications in neuroscience. Establishment of large-scale production in mammalian adherent and suspension cultures has allowed production of hundreds of milligrams of membrane proteins that has allowed their submission to serious structural biology approaches. In this

  2. 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.

  3. Expression of nuclear membrane proteins in normal, hyperplastic, and neoplastic thyroid epithelial cells.

    PubMed

    Wang, Jieying; Kondo, Tetsuo; Yamane, Tetsu; Nakazawa, Tadao; Oish, Naoki; Mochizuki, Kunio; Katoh, Ryohei

    2015-10-01

    Emerin, lamin A/C, lamin B, and lamin-associated polypeptide 2 (LAP2) are nuclear membrane proteins that play an important role in maintaining nuclear structure and coordinating cell activity. We studied the expression and significance of nuclear membrane proteins in neoplastic thyroid cells by immunohistochemistry, RT-PCR, and real-time PCR. In papillary carcinomas (PCs), the nuclear proteins most frequently expressed at high levels were emerin (82 % positive), lamin A/C (64 %), and LAP2 (82 %). Follicular carcinomas (FCs) most frequently expressed lamin B, while none of the undifferentiated carcinomas (UCs) showed strong expression of emerin or lamin A/C. In all medullary carcinomas (MCs), intermediate to high levels of expression of lamin A/C and LAP2 were found. By RT-PCR analysis, messenger RNA (mRNA) expression of all nuclear membrane proteins except emerin was higher in PC than in normal tissue. Real-time PCR analysis showed that mRNA expression of nuclear membrane protein varied between cell lines. Our findings suggest that expression of nuclear membrane proteins may be related to follicular function in normal and hyperplastic follicles, and we hypothesize that they are also involved in the proliferation and differentiation of neoplastic thyroid cells. We suggest that they reflect the biological nature and/or function of normal, hyperplastic, and neoplastic thyroid cells and may have some value in diagnosing thyroid tumors.

  4. 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

  5. Practical aspects in expression and purification of membrane proteins for structural analysis.

    PubMed

    Vinothkumar, Kutti R; Edwards, Patricia C; Standfuss, Joerg

    2013-01-01

    A surge of membrane protein structures in the last few years can be attributed to advances in technologies starting at the level of genomes, to highly efficient expression systems, stabilizing conformational flexibility, automation of crystallization and data collection for screening large numbers of crystals and the microfocus beam lines at synchrotrons. The substantial medical importance of many membrane proteins provides a strong incentive to understand them at the molecular level. It is becoming obvious that the major bottleneck in many of the membrane projects is obtaining sufficient amount of stable functional proteins in a detergent micelle for structural studies. Naturally, large effort has been spent on optimizing and advancing multiple expression systems and purification strategies that have started to yield sufficient protein and structures. We describe in this chapter protocols to refold membrane proteins from inclusion bodies, purification from inner membranes of Escherichia coli and from mammalian cell lines.

  6. 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.

  7. Science review: Cell membrane expression (connectivity) regulates neutrophil delivery, function and clearance

    PubMed Central

    Seely, Andrew JE; Pascual, José L; Christou, Nicolas V

    2003-01-01

    As the principal cellular component of the inflammatory host defense and contributor to host injury after severe physiologic insult, the neutrophil is inherently coupled to patient outcome in both health and disease. Extensive research has focused on the mechanisms that regulate neutrophil delivery, function, and clearance from the inflammatory microenvironment. The neutrophil cell membrane mediates the interaction of the neutrophil with the extracellular environment; it expresses a complex array of adhesion molecules and receptors for various ligands, including mediators, cytokines, immunoglobulins, and membrane molecules on other cells. This article presents a review and analysis of the evidence that the neutrophil membrane plays a central role in regulating neutrophil delivery (production, rolling, adhesion, diapedesis, and chemotaxis), function (priming and activation, microbicidal activity, and neutrophil-mediated host injury), and clearance (apoptosis and necrosis). In addition, we review how change in neutrophil membrane expression is synonymous with change in neutrophil function in vivo. Employing a complementary analysis of the neutrophil as a complex system, neutrophil membrane expression may be regarded as a measure of neutrophil connectivity, with altered patterns of connectivity representing functionally distinct neutrophil states. Thus, not only does the neutrophil membrane mediate the processes that characterize the neutrophil lifecycle, but characterization of neutrophil membrane expression represents a technology with which to evaluate neutrophil function. PMID:12930553

  8. 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.

  9. 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

  10. Prominent expression of xenobiotic efflux transporters in mouse extraembryonic fetal membranes compared with placenta.

    PubMed

    Aleksunes, Lauren M; Cui, Yue; Klaassen, Curtis D

    2008-09-01

    Fetal exposure to xenobiotics can be restricted by transporters at the interface between maternal and fetal circulation. Previous work identified transporters in the placenta; however, less is known about the presence of these transporters in the fetal membranes (i.e., yolk sac and amniotic membranes). The purpose of this study was to quantify mRNA and protein expression of xenobiotic transporters in mouse placenta and fetal membranes during mid to late gestation. Concepti (placenta and fetal membranes, gestation day 11) or placenta and fetal membranes (gestation days 14 and 17) were collected from pregnant mice and analyzed for expression of multidrug resistance-associated proteins (Mrps), multidrug resistance proteins (Mdrs), multidrug and toxin extrusion proteins (Mates), breast cancer resistance protein (Bcrp), and organic anion-transporting polypeptides (Oatps). Maternal liver and kidneys were also collected at day 14 for mRNA and immunohistochemical analysis. mRNA expression of Mrp, Mdr, Bcrp, Mate-1, and Oatp isoforms was detected at day 11. The uptake carriers Oatp2a1, 3a1, 4a1, and 5a1 showed placenta-predominant expression. At days 14 and 17, fetal membranes expressed higher mRNA levels of the efflux transporters Mrp2 (7-fold), Mrp4 (5-fold), Mrp5 (3-fold), Mrp6 (12-fold), Bcrp (2-fold), and Mate-1 (7-fold) than placenta. Western blot analysis of Mrp2, Mrp4, Mrp6, and Bcrp confirmed higher expression in fetal membranes. Immunostaining revealed apical (Mrp2 and Bcrp) and basolateral (Mrp4, 5, and 6) cellular localization in epithelial cells of the yolk sac. In conclusion, xenobiotic transporters in the fetal membranes may provide an additional route to protect the fetus against endogenous chemicals and xenobiotics.

  11. Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide.

    PubMed

    Deb, Arpan; Johnson, William A; Kline, Alexander P; Scott, Boston J; Meador, Lydia R; Srinivas, Dustin; Martin-Garcia, Jose M; Dörner, Katerina; Borges, Chad R; Misra, Rajeev; Hogue, Brenda G; Fromme, Petra; Mor, Tsafrir S

    2017-01-01

    Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.

  12. Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide

    PubMed Central

    Deb, Arpan; Johnson, William A.; Kline, Alexander P.; Scott, Boston J.; Meador, Lydia R.; Srinivas, Dustin; Martin-Garcia, Jose M.; Dörner, Katerina; Borges, Chad R.; Misra, Rajeev; Hogue, Brenda G.; Fromme, Petra

    2017-01-01

    Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models. PMID:28225803

  13. Mimicking photosynthetic solar energy transduction.

    PubMed

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

    2001-01-01

    Increased understanding of photosynthetic energy conversion and advances in chemical synthesis and instrumentation have made it possible to create artificial nanoscale devices and semibiological hybrids that carry out many of the functions of the natural process. Artificial light-harvesting antennas can be synthesized and linked to artificial reaction centers that convert excitation energy to chemical potential in the form of long-lived charge separation. Artificial reaction centers can form the basis for molecular-level optoelectronic devices. In addition, they may be incorporated into the lipid bilayer membranes of artificial vesicles, where they function as components of light-driven proton pumps that generate transmembrane proton motive force. The proton gradient may be used to synthesize adenosine triphosphate via an ATP synthase enzyme. The overall energy transduction process in the liposomal system mimics the solar energy conversion system of a photosynthetic bacterium. The results of this research illustrate the advantages of designing functional nanoscale devices based on biological paradigms.

  14. Genetic selection system for improving recombinant membrane protein expression in E. coli

    PubMed Central

    Massey-Gendel, Elizabeth; Zhao, Anni; Boulting, Gabriella; Kim, Hye-Yeon; Balamotis, Michael A; Seligman, Len M; Nakamoto, Robert K; Bowie, James U

    2009-01-01

    A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold. PMID:19165721

  15. 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

    2015-01-06

    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.

  16. Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

    PubMed

    de Hoog, Hans-Peter M; Lin JieRong, Esther M; Banerjee, Sourabh; Décaillot, Fabien M; Nallani, Madhavan

    2014-01-01

    G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.

  17. 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.

  18. 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

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

    PubMed Central

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

    2002-01-01

    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 γ-aminobutyric acid, α-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. PMID:12237406

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. Expression of cyclo-oxygenase types-1 and -2 in human fetal membranes throughout pregnancy.

    PubMed

    Slater, D; Dennes, W; Sawdy, R; Allport, V; Bennett, P

    1999-04-01

    Human labour is associated with increased prostaglandin synthesis within the fetal membranes. We have studied the expression of the two isoforms of the central prostaglandin synthetic enzyme, cyclo-oxygenase (COX-1 and COX-2), in human fetal membranes throughout pregnancy, at mRNA, protein and activity levels. COX-1 mRNA expression was low in human amnion and chorion-decidua and did not change with gestational age. COX-2 mRNA expression in fetal membranes increased with gestational age, with significant up-regulation prior to the onset of labour and in association with labour. Protein concentrations of COX-1 did not change, whilst concentrations of COX-2 increased from the first to the third trimester. COX activity increased with gestational age and in association with labour, although prostaglandin production in fetal membranes collected after labour was reduced, suggesting reduced substrate supply. These data suggest that it is up-regulation of COX-2, rather than of COX-1, which mediates increased prostaglandin synthesis within the fetal membranes at term. Much of the increase in COX-2 expression precedes the onset of labour, suggesting that it is a cause, rather than a consequence, of labour.

  5. 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...

  6. 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.

  7. The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis.

    PubMed

    Morini, Marina; Peñaranda, David S; Vílchez, M Carmen; Tveiten, Helge; Lafont, Anne-Gaëlle; Dufour, Sylvie; Pérez, Luz; Asturiano, Juan F

    2017-01-01

    Estradiol (E2) can bind to nuclear estrogen receptors (ESR) or membrane estrogen receptors (GPER). While mammals possess two nuclear ESRs and one membrane GPER, the European eel, like most other teleosts, has three nuclear ESRs and two membrane GPERs, as the result of a teleost specific genome duplication. In the current study, the expression of the three nuclear ESRs (ESR1, ESR2a and ESR2b) and the two membrane GPERs (GPERa and GPERb) in the brain-pituitary-gonad (BPG) axis of the European eel was measured, throughout spermatogenesis. The eels were first transferred from freshwater (FW) to seawater (SW), inducing parallel increases in E2 plasma levels and the expression of ESRs. This indicates that salinity has a stimulatory effect on the E2 signalling pathway along the BPG axis. Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E2 plasma levels, and different patterns of expression of ESRs and GPERs in the BPG axis. The expression of nuclear ESRs increased in some parts of the brain, suggesting a possible upregulation due to a local production of E2. In the testis, the highest expression levels of the nuclear ESRs were observed at the beginning of spermatogenesis, possibly mediating the role of E2 as spermatogonia renewal factor, followed by a sharply decrease in the expression of ESRs. Conversely, there was a marked increase observed in the expression of both membrane GPERs throughout spermatogenesis, suggesting they play a major role in the final stages of spermatogenesis.

  8. Importance of luminal membrane mesothelin expression in intraductal papillary mucinous neoplasms.

    PubMed

    Einama, Takahiro; Kamachi, Hirofumi; Nishihara, Hiroshi; Homma, Shigenori; Kanno, Hiromi; Ishikawa, Marin; Kawamata, Futoshi; Konishi, Yuji; Sato, Masanori; Tahara, Munenori; Okada, Kuniaki; Muraoka, Shunji; Kamiyama, Toshiya; Taketomi, Akinobu; Matsuno, Yoshihiro; Furukawa, Hiroyuki; Todo, Satoru

    2015-04-01

    The present study demonstrated that luminal membrane mesothelin expression is a reliable prognostic factor in gastric cancer. Intraductal papillary mucinous neoplasms (IPMNs) often exhibit a spectrum of dysplasia, ranging between adenoma and carcinoma. Therefore, an immunohistochemical analysis of mesothelin expression in IPMN was performed in the present study, focusing on the localization of mesothelin. IPMNs were classified into two groups, IPMNs associated with invasive carcinoma and low-high (L-H) grade dysplasias. The tumors were classified as mesothelin-positive or -negative and in the mesothelin-positive cases, the localization of mesothelin was evaluated as luminal membrane- or cytoplasmic-positive. Among the 37 IPMNs, mesothelin expression was observed in 21 samples (56.8%), including 46.2% (12 out of 26) of the L-H dysplasia and 81.8% (9 out of 11) of the invasive carcinoma samples (P=0.071). Luminal membrane localization was observed in 10 samples (27%), including 15.4% (4/26) of the L-H dysplasia samples and 54.5% (6 out of 11) of the invasive carcinoma samples (P=0.022). Six patients experienced post-operative recurrence, with five of the recurrent tumors exhibiting mesothelin expression and all six exhibiting luminal membrane localization. It was concluded that immunohistochemical examinations for mesothelin expression and localization are clinically useful for prognostic assessments and decision making regarding further treatment subsequent to surgical procedures in patients with IPMN.

  9. Comparison of Gene Expression Profile of Epiretinal Membranes Obtained from Eyes with Proliferative Vitreoretinopathy to That of Secondary Epiretinal Membranes

    PubMed Central

    Asato, Ryo; Yoshida, Shigeo; Ogura, Atsushi; Nakama, Takahito; Ishikawa, Keijiro; Nakao, Shintaro; Sassa, Yukio; Enaida, Hiroshi; Oshima, Yuji; Ikeo, Kazuho; Gojobori, Takashi; Kono, Toshihiro; Ishibashi, Tatsuro

    2013-01-01

    Background Proliferative vitreoretinopathy (PVR) is a destructive complication of retinal detachment and vitreoretinal surgery which can lead to severe vision reduction by tractional retinal detachments. The purpose of this study was to determine the gene expression profile of epiretinal membranes (ERMs) associated with a PVR (PVR-ERM) and to compare it to the expression profile of less-aggressive secondary ERMs. Methodology/Principal Findings A PCR-amplified complementary DNA (cDNA) library was constructed using the RNAs isolated from ERMs obtained during vitrectomy. The sequence from the 5′ end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). We obtained 1116 nonredundant clusters representing individual genes expressed in PVR-ERMs, and 799 clusters representing the genes expressed in secondary ERMs. The transcriptome of the PVR-ERMs was subdivided by functional subsets of genes related to metabolism, cell adhesion, cytoskeleton, signaling, and other functions, by FatiGo analysis. The genes highly expressed in PVR-ERMs were compared to those expressed in the secondary ERMs, and these were subdivided by cell adhesion, proliferation, and other functions. Querying 10 cell adhesion-related genes against the STRING database yielded 70 possible physical relationships to other genes/proteins, which included an additional 60 genes that were not detected in the PVR-ERM library. Of these, soluble CD44 and soluble vascular cellular adhesion molecule-1 were significantly increased in the vitreous of patients with PVR. Conclusions/Significance Our results support an earlier hypothesis that a PVR-ERM, even from genomic points of view, is an aberrant form of wound healing response. Genes preferentially expressed in PVR-ERMs may play an important role in the progression of PVR and could be served as therapeutic targets. PMID:23372684

  10. Production of Computationally Designed Small Soluble- and Membrane-Proteins: Cloning, Expression, and Purification.

    PubMed

    Tripathy, Barsa; Acharya, Rudresh

    2017-01-01

    This book chapter focuses on expression and purification of computationally designed small soluble proteins and membrane proteins that are ordinarily difficult to express in good amounts for experiments. Over-expression of such proteins can be achieved by using the solubility tag such as maltose binding protein (MBP), Thioredoxin (Trx), and Gultathione-S-transferase (GST) fused to the protein of interest. Here, we describe and provide the protocols for cloning, expression and purification of such proteins using the solubility tag.

  11. Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins

    DTIC Science & Technology

    2005-01-01

    with Brucella melitensis WR201(16MDeltapurEK), immunized intramuscularly with dialyzed cell lysate of Infect. Immun. 67 (1999) 5877-5884. B. melitensis ...bacterioferritin gene of Brucella melitensis 16M strain, FEBS Lett. 361 (2-3) (1995) 238-242. serum and derived IgG had strong reaction to the Bru- [7] L.E. Lindler...detection by the antiserum. The pro- nucleotide sequence, and expression of the Brucella melitensis tein samples were prepared by treatment of WRR51 omp31

  12. 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

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

    PubMed

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

    2005-10-01

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

  14. 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.

  15. 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...

  16. In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS).

    PubMed

    Krupka, Simone S; Wiltschi, Birgit; Reuning, Ute; Hölscher, Kerstin; Hara, Masahiko; Sinner, Eva-Kathrin

    2006-08-15

    Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.

  17. Fibroblast Circadian Rhythms of PER2 Expression Depend on Membrane Potential and Intracellular Calcium

    PubMed Central

    Noguchi, Takako; Wang, Connie W.; Pan, Haiyun

    2012-01-01

    The suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes circadian rhythms of cells and tissues throughout the body. In SCN neurons, rhythms of clock gene expression are suppressed by manipulations that hyperpolarize the plasma membrane or lower intracellular Ca2+. However, whether clocks in other cells also depend on membrane potential and calcium is unknown. In this study, we investigate the effects of membrane potential and intracellular calcium on circadian rhythms in mouse primary fibroblasts. Rhythms of clock gene expression were monitored using a PER2::LUC knockin reporter. We found that rhythms were lost or delayed at lower (hyperpolarizing) K+ concentrations. Bioluminescence imaging revealed that this loss of rhythmicity in cultures was due to loss of rhythmicity of single cells rather than desynchrony among cells. In lower Ca2+ concentrations, rhythms were advanced or had shorter periods. Buffering intracellular Ca2+ by the calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) or manipulation of IP3-sensitive intracellular calcium stores by thapsigargin delayed rhythms. These results suggest that the circadian clock in fibroblasts, as in SCN neurons, is regulated by membrane potential and Ca2+. Changes in intracellular Ca2+ may mediate the effects of membrane potential that we observed. PMID:22734566

  18. Fibroblast circadian rhythms of PER2 expression depend on membrane potential and intracellular calcium.

    PubMed

    Noguchi, Takako; Wang, Connie W; Pan, Haiyun; Welsh, David K

    2012-07-01

    The suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes circadian rhythms of cells and tissues throughout the body. In SCN neurons, rhythms of clock gene expression are suppressed by manipulations that hyperpolarize the plasma membrane or lower intracellular Ca(2+). However, whether clocks in other cells also depend on membrane potential and calcium is unknown. In this study, the authors investigate the effects of membrane potential and intracellular calcium on circadian rhythms in mouse primary fibroblasts. Rhythms of clock gene expression were monitored using a PER2::LUC knockin reporter. Rhythms were lost or delayed at lower (hyperpolarizing) K(+) concentrations. Bioluminescence imaging revealed that this loss of rhythmicity in cultures was due to loss of rhythmicity of single cells rather than loss of synchrony among cells. In lower Ca(2+) concentrations, rhythms were advanced or had shorter periods. Buffering intracellular Ca(2+) by the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) or manipulation of inositol triphosphate (IP(3))-sensitive intracellular calcium stores by thapsigargin delayed rhythms. These results suggest that the circadian clock in fibroblasts, as in SCN neurons, is regulated by membrane potential and Ca(2+). Changes in intracellular Ca(2+) may mediate the effects of membrane potential observed in this study.

  19. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    PubMed Central

    2011-01-01

    Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP. PMID:21929758

  20. Mycorrhizal fungi influence on silver uptake and membrane protein gene expression following silver nanoparticle exposure

    NASA Astrophysics Data System (ADS)

    Noori, Azam; White, Jason C.; Newman, Lee A.

    2017-02-01

    The rapid growth of nanotechnology and the high demand for nanomaterial use have greatly increased the risk of particle release into the environment. Understanding nanomaterial interactions with crop species and their associated microorganisms is critical to food safety and security. In the current study, tomato was inoculated with mycorrhizal fungi and subsequently exposed to 12, 24, or 36 mg/kg of 2- or 15-nm silver nanoparticles (Ag-NPs). Mycorrhizal (M) and non-mycorrhizal (NM) tomatoes exposed to 36 mg/kg of 2-nm Ag-NPs accumulated 1300 and 1600 μg/g silver in their tissues, respectively. Mycorrhizal plants accumulated 14% less silver compared to non-mycorrhizal plants. To begin to understand the mechanisms by which plants accumulate NPs, the expression of two aquaporin channel genes, the plasma membrane intrinsic protein (PIP) and the tonoplast membrane intrinsic protein (TIP), and one potassium channel (KC) gene were studied. In non-mycorrhizal plants, the expression of KC, PIP, and TIP was eight, five, and nine times higher than the control, respectively. These expressions for mycorrhizal plants were 5.8, 3.5, and 2 times higher than controls, respectively. The expression of KC and PIP, which are located on the plasma membrane, was 3.5 and 2.5, respectively, times higher than TIP, which is located on the tonoplast. PIP expression was significantly higher in NM tomatoes exposed to 12 mg/kg of 2-nm Ag-NPs compared to M plants. These results show that mycorrhizal colonization decreases Ag accumulation in NP-exposed plants and also moderates changes in expression level of membrane transport proteins.

  1. Photosynthetic system in Blastochloris viridis revisited.

    PubMed

    Konorty, Marina; Brumfeld, Vlad; Vermeglio, Andre; Kahana, Nava; Medalia, Ohad; Minsky, Abraham

    2009-06-09

    The bacterium Blastochloris viridis carries one of the simplest photosynthetic systems, which includes a single light-harvesting complex that surrounds the reaction center, membrane soluble quinones, and a soluble periplasmic protein cytochrome c(2) that shuttle between the reaction center and the bc(1) complex and act as electron carriers, as well as the ATP synthase. The close arrangement of the photosynthetic membranes in Bl. viridis, along with the extremely tight arrangement of the photosystems within these membranes, raises a fundamental question about the diffusion of the electron carriers. To address this issue, we analyzed the structure and response of the Bl. viridis photosynthetic system to various light conditions, by using a combination of electron microscopy, whole-cell cryotomography, and spectroscopic methods. We demonstrate that in response to high light intensities, the ratio of both cytochrome c(2) and bc(1) complexes to the reaction centers is increased. The shorter membrane stacks, along with the notion that the bc(1) complex is located at the highly curved edges of these stacks, result in a smaller average distance between the reaction centers and the bc(1) complexes, leading to shorter pathways of cytochrome c(2) between the two complexes. Under anaerobic conditions, the slow diffusion rate is further mitigated by keeping most of the quinone pool reduced, resulting in a concentration gradient of quinols that allows for a constant supply of theses electron carriers to the bc(1) complex.

  2. Photosynthetic reaction center complexes from heliobacteria

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

    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 of this project is the family of newly discovered strictly anaerobic photosynthetic organisms known as Heliobacteria. These organisms are the only known photosynthetic organisms that are grouped with the gram-positive phylum of bacteria. The properties of these reaction centers suggest that they might be the decendants of an ancestor that also gave rise to Photosystem 1 found in oxygen-evolving photosynthetic organisms. Photoactive reaction center-core antenna complexes have been isolated from the photosynthetic bacteria Heliobacillus mobilis and Heliobacterium gestii. The absorption and fluorescence properties of membranes and reaction centers are almost identical, suggesting that a single pigment-protein complex serves as both antenna and reaction center. Experiments in progress include sequence determination of the 48,000 Mr reaction center protein, and evolutionary comparisons with other reaction center proteins.

  3. 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.

  4. 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.

  5. 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.

  6. Expression of a Translationally Fused TAP-Tagged Plasma Membrane Proton Pump in Arabidopsis thaliana

    PubMed Central

    2015-01-01

    The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report the translational fusion of a tandem affinity-purification tag to the 5′ end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryonic lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS-PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 functions and have shown that these plants can be used to purify AHA1 protein and to identify in planta interacting proteins by mass spectrometry. PMID:24397334

  7. Cloning, expression, purification, and characterization of the membrane protein UncI from Escherichia coli.

    PubMed

    Hartmann, Claudia; Engel, Andreas

    2011-10-01

    The Escherichia coli unc-operon encodes the genes for the subunits of the F0F1-ATP synthase and an integral membrane protein of unknown function called UncI. UncI influences the cell-growth and activity of F0F1, but its exact function is still unknown. The expression level is too low to extract milligram amounts of UncI from E. coli membranes and the existing purification protocol based on methanol/chloroform is not suitable for structural and functional studies. Here we present protocols to increase the expression level, to purify UncI in a detergent where UncI is monodisperse, and we characterize its oligomeric state.

  8. Plasma Membrane Expression of Heat Shock Protein 60 In Vivo in Response to Infection

    PubMed Central

    Belles, Cindy; Kuhl, Alicia; Nosheny, Rachel; Carding, Simon R.

    1999-01-01

    Heat shock protein 60 (hsp60) is constitutively expressed in the mitochondria of eukaryotic cells. However, it has been identified in other subcellular compartments in several disease states and in transformed cells, and it is an immunogenic molecule in various infectious and autoimmune diseases. To better understand the factors that influence expression of hsp60 in normal cells in vivo, we analyzed its cellular and subcellular distribution in mice infected with the intracellular bacterium Listeria monocytogenes. Western blotting of subcellular fractionated spleen cells showed that although endogenous hsp60 was restricted to the mitochondria in noninfected animals, it was associated with the plasma membrane as a result of infection. The low levels of plasma membrane-associated hsp60 seen in the livers in noninfected animals subsequently increased during infection. Plasma membrane hsp60 expression did not correlate with bacterial growth, being most evident during or after bacterial clearance and persisting at 3 weeks postinfection. Using flow cytometry, we determined that Mac-1+, T-cell receptor γδ+, and B220+ cells represented the major Hsp60+ populations in spleens of infected mice. By contrast, B220+ cells were the predominant hsp60+ population in livers of infected mice. Of the immune cells analyzed, the kinetic profile of the γδ T-cell response most closely matched that of hsp60 expression in both the spleen and liver. Collectively, these findings show that during infection hsp60 can be localized to the plasma membrane of viable cells, particularly antigen-presenting cells, providing a means by which hsp60-reactive lymphocytes seen in various infectious disease and autoimmune disorders may be generated and maintained. PMID:10417191

  9. 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.

  10. Osteopontin expression in vitreous and proliferative retinal membranes of patients with proliferative vitreous retinopathy

    PubMed Central

    Liu, Xiao-Yi; Li, Lei; Yao, Jia-Qi; Chen, Xi; Liu, Qing-Huai

    2011-01-01

    AIM To analyze osteopontin (OPN) expression in vitreous and proliferative retinal membranes of patients with proliferative vitreous retinopathy (PVR). METHODS A total of 54 vitreous fluid samples were obtained between 2009 and 2010, which contained 45 with PVR (group A) and 9 without PVR (group B). Enzyme-linked immunosorbent assay was applied to quantify the OPN concentrations in vitreous fluid. Four samples of proliferative retinal membrane were also obtained at the time of vitrectomy, and their contents of OPN were measured by Real-time RT-PCR. RESULTS The OPN levels in the vitreous fluid were 778.48±62.06ng/mL in group A and 452.99±32.52ng/mL in group B. The vitreous OPN levels in group A were significantly higher than those in group B and to rise by time in the early stages of PVR. The average OPN levels in the proliferative retinal membranes (F=0.14) were also higher than those in the retinal pigment cells (F=0) using Real-time RT-PCR. CONCLUSION The high vitreous and proliferative retinal membrane OPN levels in PVR suggest that OPN might promote the development of PVR. The vitreous OPN concentrations are rising by the time in the early phases of PVR. PMID:22553691

  11. Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo

    PubMed Central

    1994-01-01

    ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed. PMID:8294513

  12. A C-terminal di-leucine motif controls plasma membrane expression of PMCA4b.

    PubMed

    Antalffy, Géza; Pászty, Katalin; Varga, Karolina; Hegedűs, Luca; Enyedi, Agnes; Padányi, Rita

    2013-12-01

    Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.

  13. Expression and Characterization of a Mycoplasma genitalium Glycosyltransferase in Membrane Glycolipid Biosynthesis

    PubMed Central

    Andrés, Eduardo; Martínez, Núria; Planas, Antoni

    2011-01-01

    Mycoplasmas contain glycoglycerolipids in their plasma membrane as key structural components involved in bilayer properties and stability. A membrane-associated glycosyltransferase (GT), GT MG517, has been identified in Mycoplasma genitalium, which sequentially produces monoglycosyl- and diglycosyldiacylglycerols. When recombinantly expressed in Escherichia coli, the enzyme was functional in vivo and yielded membrane glycolipids from which Glcβ1,6GlcβDAG was identified as the main product. A chaperone co-expression system and extraction with CHAPS detergent afforded soluble protein that was purified by affinity chromatography. GT MG517 transfers glucosyl and galactosyl residues from UDP-Glc and UDP-Gal to dioleoylglycerol (DOG) acceptor to form the corresponding β-glycosyl-DOG, which then acts as acceptor to give β-diglycosyl-DOG products. The enzyme (GT2 family) follows Michaelis-Menten kinetics. kcat is about 5-fold higher for UDP-Gal with either DOG or monoglucosyldioleoylglycerol acceptors, but it shows better binding for UDP-Glc than UDP-Gal, as reflected by the lower Km, which results in similar kcat/Km values for both donors. Although sequentially adding glycosyl residues with β-1,6 connectivity, the first glycosyltransferase activity (to DOG) is about 1 order of magnitude higher than the second (to monoglucosyldioleoylglycerol). Because the ratio between the non-bilayer-forming monoglycosyldiacylglycerols and the bilayer-prone diglycosyldiacylglycerols contributes to regulate the properties of the plasma membrane, both synthase activities are probably regulated. Dioleoylphosphatidylglycerol (anionic phospholipid) activates the enzyme, kcat linearly increasing with dioleoylphosphatidylglycerol concentration. GT MG517 is shown to be encoded by an essential gene, and the addition of GT inhibitors results in cell growth inhibition. It is proposed that glycolipid synthases are potential targets for drug discovery against infections by mycoplasmas. PMID

  14. Expression of BMP and Actin Membrane Bound Inhibitor Is Increased during Terminal Differentiation of MSCs

    PubMed Central

    Karl, Alexandra; Berner, Arne; Schmitz, Paul; Koch, Matthias; Nerlich, Michael; Mueller, Michael B.

    2016-01-01

    Chondrogenic differentiating mesenchymal stem cells (MSCs) are mimicking embryonal endochondral ossification and become hypertrophic. BMP (bone morphogenetic protein) and Activin Membrane Bound Inhibitor (BAMBI) is a pseudoreceptor that regulates the activity of transforming growth factor-β (TGF-β) and BMP signalling during chondrogenesis. Both TGF-β and BMP signalling are regulators of chondrogenic cell differentiation. Human bone marrow derived MSCs were chondrogenically predifferentiated in aggregate culture for 14 days. Thereafter, one group was subjected to hypertrophy enhancing media conditions while controls were kept in chondrogenic medium until day 28. Histological evaluation, gene expression by PCR, and Western blot analysis were carried out at days 1, 3, 7, 14, 17, 21, and 28. A subset of cultures was treated with the BMP inhibitor Noggin to test for BMP dependent expression of BAMBI. Hypertrophic differentiated pellets showed larger cells with increased collagen 10 and alkaline phosphatase staining. There was significantly increased expression of BAMBI on gene expression and protein level in hypertrophic cultures compared to the chondrogenic control and increased BMP4 gene expression. Immunohistochemistry showed intense staining of BAMBI in hypertrophic cells. BAMBI expression was dose-dependently downregulated by Noggin. The pseudoreceptor BAMBI is upregulated upon enhancement of hypertrophy in MSC chondrogenic differentiation by a BMP dependent mechanism. PMID:27843458

  15. Expression and function of membrane regulators of complement on rat astrocytes in culture.

    PubMed Central

    Rogers, C A; Gasque, P; Piddlesden, S J; Okada, N; Holers, V M; Morgan, B P

    1996-01-01

    Human astrocytes express CD59, decay accelerating factor and membrane cofactor protein to restrict the damaging effect of complement (C) activation on their cell surface. 5I2 antigen (5I2 Ag) is the functional analogue of the latter two proteins in rats. We here demonstrate the surface expression on rat astrocytes of CD59 and 5I2 Ag and use sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting to confirm their identity and to quantify expression. Rat CD59 (MW 20,000) was expressed at 720 x 10(3) molecules per cell and 5I2 Ag (MW 58,000 and 64,000) at 625 x 10(3) molecules per cell. Reverse transcription-polymerase chain reaction using specific oligonucleotide primers demonstrated expression of mRNA for each protein. Twenty-four-hour stimulation with inflammatory cytokines (interferon-gamma, tumour necrosis factor-alpha, interleukins-1 beta, -2 and -6) or phorbol myristate acetate had no significant effect on the level of expression of either protein as determined by Western blotting. Lysis caused by classical pathway activation of C in human or rat serum was enhanced by blocking the function of CD59 and 5I2 Ag on rat astrocytes with monoclonal antibodies. Images Figure 2 Figure 3 Figure 4 PMID:8707343

  16. Photosynthetic characteristics of an amphibious plant, Eleocharis vivipara: Expression of C sub 4 and C sub 3 modes in contrasting environments

    SciTech Connect

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

    1988-09-01

    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 C{sub 4} and C{sub 3} photosynthesis. In the terrestrial form, the culms, which are photosynthetic organs, possess a Kranz-type anatomy typical of C{sub 4} plants, and well-developed bundle-sheath cells contain numerous large chloroplasts. In the submersed form, the culms possess anatomical features characteristic of submersed aquatic plants, and the reduced bundle-sheath cells contain only a few small chloroplasts. {sup 14}C pulse-{sup 12}C chase experiments showed that the terrestrial form and the submersed form fix carbon by way of the C{sub 4} pathway, with aspartate (40%) and malate (35%) as the main primary products, and by way of the C{sub 3} 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-C{sub 4} subtype, whereas the submersed form showed decreased activities of key C{sub 4} enzymes and an increased ribulose 1,5-bisphosphate carboxylase activity. These data suggest that this species can differentiate into the C{sub 4} mode under terrestrial conditions and into the C{sub 3} mode under submersed conditions.

  17. 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

    2014-12-05

    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.

  18. Expression, detergent solubilization, and purification of a membrane transporter, the MexB multidrug resistance protein.

    PubMed

    Bhatt, Forum H; Jeffery, Constance J

    2010-12-03

    Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP). The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC). In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

  19. Increased expression of the integral membrane proteins EGFR and FGFR3 in anti-apoptotic Chinese hamster ovary cell lines.

    PubMed

    Ohsfeldt, Erika; Huang, Szu-Han; Baycin-Hizal, Deniz; Kristoffersen, Linda; Le, Thuy-My T; Li, Edwin; Hristova, Kalina; Betenbaugh, Michael J

    2012-01-01

    Membrane proteins such as receptor tyrosine kinases (RTKs) have a vital role in many cellular functions, making them potential targets for therapeutic research. In this study, we investigated the coexpression of the anti-apoptosis gene Bcl-x(L) with model membrane proteins as a means of increasing membrane protein expression in mammalian cells. Chinese hamster ovary (CHO) cells expressing heterologous Bcl-x(L) and wild-type CHO cells were transfected with either epidermal growth factor receptor or fibroblast growth factor receptor 3. The CHO-Bcl-x(L) cell lines showed increased expression of both RTK proteins as compared with the wild-type CHO cell lines in transient expression analysis, as detected by Western blot and flow cytometry after 15 days of antibiotic selection in stable expression pools. Increased expression was also seen in clonal isolates from the CHO-Bcl-x(L) cell lines, whereas the clonal cell line expression was minimal in wild-type CHO cell lines. Our results demonstrate that application of the anti-apoptosis gene Bcl-x(L) can increase expression of RTK proteins in CHO cells. This approach may be applied to improve stable expression of other membrane proteins in the future using mammalian cell lines with Bcl-x(L) or perhaps other anti-apoptotic genes.

  20. Gene Expression Analysis of the Irrigation Solution Samples Collected during Vitrectomy for Idiopathic Epiretinal Membrane

    PubMed Central

    Myojin, Sayaka; Yoshida, Shigeo; Takeda, Atsunobu; Murakami, Yusuke; Kawano, Yoichi; Oshima, Yuji; Ishibashi, Tatsuro; Sonoda, Koh-Hei

    2016-01-01

    Purpose The analysis of gene expression in idiopathic epiretinal membranes (iERMs) may help elucidate ERM formation and its pathology. Here, we conducted a case-control study, in order to determine the expression levels of cytokines and other genes in eyes with macular hole (MH) or iERM. Methods Twenty eyes, obtained from seven male and 13 female patients, were included in the study. The average age of the study subjects was 69.1 ± 7.67 years, and 15 eyes had iERM, while five eyes had MH. Irrigation solution samples were collected during vitrectomy, centrifuged, and the levels of cytokine and other mRNAs in the sediment were assessed using real-time PCR. The expression level of 11 cytokine genes, four transcription factor genes, two cytoskeletal genes, and genes encoding two extracellular matrix proteins in eyes with MH or iERM were determined and compared. Results The expression levels of interleukin 6 (IL6), tumor growth factor B2 (TGFB2), vascular endothelial growth factor A (VEGFA), chemokine C-X-C motif ligand 1 (CXCL1), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), glial fibrillary acidic protein (GFAP), and tenascin C (TNC) were significantly higher in eyes with iERM than in eyes with MH. The expression of these genes was not associated with the preoperative visual acuity of the investigated patients. Conclusions The obtained results indicate that real-time PCR analysis of irrigation solution samples collected during vitrectomy can help assess the expression levels of several genes, and that iERM is associated with the expression of pro-inflammatory genes and the genes expressed during angiogenesis and wound healing process (IL6, TGFB2, VEGFA, CXCL1, RELA, GFAP, and TNC). PMID:27736918

  1. Cell-Specific Expression of Plasma Membrane Calcium ATPase Isoforms in Retinal Neurons

    PubMed Central

    Krizaj, David; Demarco, Steven J.; Johnson, Juliette; Strehler, Emanuel E.; Copenhagen, David R.

    2007-01-01

    Ca2+ extrusion by high-affinity plasma membrane calcium ATPases (PMCAs) is a principal mechanism for the clearance of Ca2+ from the cytosol. The PMCA family consists of four isoforms (PMCA1–4). Little is known about the selective expression of these isoforms in brain tissues or about the physiological function conferred upon neurons by any given isoform. We investigated the cellular and subcellular distribution of PMCA isoforms in a mammalian retina. Mouse photoreceptors, cone bipolar cells and horizontal cells, which respond to light with a graded polarization, express isoform 1 (PMCA1) of the PMCA family. PMCA2 is localized to rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and PMCA3 is predominantly expressed in spiking neurons, including both amacrine and ganglion cells but is also found in horizontal cells. PMCA4 was found to be selectively expressed in both synaptic layers. Optical measurements of Ca2+ clearance showed that PMCAs mediate Ca2+ extrusion in both rod and cone bipolar cells. In addition, we found that rod bipolar cells, but not cone bipolar cells possess a prominent Na+/Ca2+ exchange mechanism. We conclude that PMCA isoforms are selectively expressed in retinal neurons and that processes of Ca2+ clearance are different in rod and cone bipolar cells. PMID:12209837

  2. Interleukin 4 induces membrane Thy-1 expression on normal murine B cells.

    PubMed Central

    Snapper, C M; Hornbeck, P V; Atasoy, U; Pereira, G M; Paul, W E

    1988-01-01

    Thy-1, a cell-surface glycoprotein of undetermined function, is expressed in relatively large amounts on mouse thymocytes, peripheral T cells, and neurons. It is widely used as a marker to distinguish peripheral T cells from B cells in mice. We show here that, in five distinct mouse strains, recombinant interleukin 4 (IL-4/B-cell stimulatory factor 1) strikingly induces membrane expression of Thy-1 on the vast majority of lipopolysaccharide (LPS)-stimulated normal murine B cells. Thy-1+ B cells are precursors for immunoglobulin-secreting cells. RNA blot analysis indicates that B cells express a Thy-1 mRNA of 1.8 kilobases, the same size as that found in T cells. Cell mixing experiments show that only cells derived from Thy-1.2+ donors express Thy-1.2, indicating that B cells expressing Thy-1 have not passively absorbed the glycoprotein from another cell source. Recombinant interferon-gamma inhibits Thy-1 induction by B cells stimulated with LPS and IL-4. Thy-1 is also induced on B cells that have been stimulated as a result of the specific activation of an IL-4-producing T-helper clone. Anti-IL-4 monoclonal antibody inhibits the induction of B-cell Thy-1 in this T-cell-B-cell interaction. Images PMID:2901096

  3. 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.

  4. 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

  5. Chondrogenic potential of subpopulations of cells expressing mesenchymal stem cell markers derived from human synovial membranes.

    PubMed

    Arufe, M C; De la Fuente, A; Fuentes, I; de Toro, F J; Blanco, F J

    2010-11-01

    In this study we analyzed the chondrogenic potential of subpopulations of mesenchymal stem cells (MSCs) derived from human synovial membranes enriched for CD73, CD106, and CD271 markers. Subpopulations of human synovial membrane MSCs enriched for CD73, CD106, and CD271 markers were isolated using a cytometry sorter and characterized by flow cytometry for MSC markers. The expression of Sox9, Nanog, and Runx2 genes by these cells was measured by reverse transcriptase-polymerase chain reaction. The chondrogenesis of each subpopulation was assessed by culturing the cells in a defined medium to produce spontaneous spheroid formation and differentiation towards chondrocyte-like cells. The examination of the spheroids by histological and immunohistochemical analyses for collagen type II (COL2), aggrecan, collagen type I (COL1), metalloprotease 13 (MMP13), and collagen type X (COLX) levels were performed to assess their chondrogenesis capacity. The adipogenesis and osteogenesis potential of each subpopulation was determined using commercial media; the resulting cells were stained with oil red O or red alizarin to test the degree of differentiation. The subpopulations had different profiles of cells positive for the MSC markers CD44, CD69, CD73, CD90, and CD105 and showed different expression levels of the genes Sox9, Nanog, and Runx2 involved in chondrogenesis, undifferentiation, and osteoblastogenesis, respectively. Immunohistochemical analysis demonstrated that COL1, COL2, COLX, MMP13, and aggrecan were expressed in the spheroids as soon as 14 days of culture. The CD271(+) subpopulation expressed the highest levels of COL2 staining compared to the other subpopulations. CD105 and Runx2 were shown by immunohistochemistry and genetic analysis to have significantly higher expression CD271(+) subpopulation than the other subpopulations. Spheroids formed from CD271-enriched and CD73-enriched MSCs from normal human synovial membranes mimic the native cartilage extracellular

  6. TNFR1 mediates increased neuronal membrane EAAT3 expression after in vivo cerebral ischemic preconditioning.

    PubMed

    Pradillo, J M; Hurtado, O; Romera, C; Cárdenas, A; Fernández-Tomé, P; Alonso-Escolano, D; Lorenzo, P; Moro, M A; Lizasoain, I

    2006-01-01

    A short ischemic event (ischemic preconditioning) can result in subsequent resistance to severe ischemic injury (ischemic tolerance). Glutamate is released after ischemia and produces cell death. It has been described that after ischemic preconditioning, the release of glutamate is reduced. We have shown that an in vitro model of ischemic preconditioning produces upregulation of glutamate transporters which mediates brain tolerance. We have now decided to investigate whether ischemic preconditioning-induced glutamate transporter upregulation takes also place in vivo, its cellular localization and the mechanisms by which this upregulation is controlled. A period of 10 min of temporary middle cerebral artery occlusion was used as a model of ischemic preconditioning in rat. EAAT1, EAAT2 and EAAT3 glutamate transporters were found in brain from control animals. Ischemic preconditioning produced an up-regulation of EAAT2 and EAAT3 but not of EAAT1 expression. Ischemic preconditioning-induced increase in EAAT3 expression was reduced by the TNF-alpha converting enzyme inhibitor BB1101. Intracerebral administration of either anti-TNF-alpha antibody or of a TNFR1 antisense oligodeoxynucleotide also inhibited ischemic preconditioning-induced EAAT3 up-regulation. Immunohistochemical studies suggest that, whereas the expression of EAAT3 is located in both neuronal cytoplasm and plasma membrane, ischemic preconditioning-induced up-regulation of EAAT3 is mainly localized at the plasma membrane level. In summary, these results demonstrate that in vivo ischemic preconditioning increases the expression of EAAT2 and EAAT3 glutamate transporters the upregulation of the latter being at least partly mediated by TNF-alpha converting enzyme/TNF-alpha/TNFR1 pathway.

  7. Targeting the Warburg Effect That Arises in Tumor Cells Expressing Membrane Type-1 Matrix Metalloproteinase*

    PubMed Central

    Sakamoto, Takeharu; Niiya, Daigo; Seiki, Motoharu

    2011-01-01

    Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for cellular adaptation to hypoxia, although its physiological roles and activation mechanisms during normoxia have not been studied sufficiently. The Warburg effect, which is a hallmark of malignant tumors that is characterized by increased activity of aerobic glycolysis, accompanies activation of HIF-1 during normoxia. Besides tumor cells that have multiple genetic and epigenetic alterations, normal macrophages also use glycolysis for ATP production by depending upon elevated HIF-1 activity even during normoxia. We recently found that activity of factor inhibiting HIF-1 (FIH-1) is specifically suppressed in macrophages by a nonproteolytic activity of membrane type-1 matrix metalloproteinase (MT1-MMP/MMP-14). Thus, MT1-MMP expressed in macrophages plays a significant role in regulating HIF-1 activity during normoxia. In the light of this finding, we examined here whether MT1-MMP contributes to the Warburg effect of tumor cells. All the tumor cell lines that express MT1-MMP exhibit increased glycolytic activity, and forced expression of MT1-MMP in MT1-MMP-negative tumor cells is sufficient to induce the Warburg effect. The cytoplasmic tail of MT1-MMP mediates the stimulation of aerobic glycolysis by increasing the expression of HIF-1 target genes. Specific intervention of the MT1-MMP-mediated activation of HIF-1 in tumor cells retarded tumor growth in mice. Systemic administration of a membrane-penetrating form of the cytoplasmic tail peptide in mice to inhibit HIF-1 activation competitively also exhibited a therapeutic effect on tumors. PMID:21372132

  8. Impact of light intensity and quality on chromatophore and nuclear gene expression in Paulinella chromatophora, an amoeba with nascent photosynthetic organelles.

    PubMed

    Zhang, Ru; Nowack, Eva C M; Price, Dana C; Bhattacharya, Debashish; Grossman, Arthur R

    2017-04-01

    Plastid evolution has been attributed to a single primary endosymbiotic event that occurred about 1.6 billion years ago (BYA) in which a cyanobacterium was engulfed and retained by a eukaryotic cell, although early steps in plastid integration are poorly understood. The photosynthetic amoeba Paulinella chromatophora represents a unique model for the study of plastid evolution because it contains cyanobacterium-derived photosynthetic organelles termed 'chromatophores' that originated relatively recently (0.09-0.14 BYA). The chromatophore genome is about a third the size of the genome of closely related cyanobacteria, but 10-fold larger than most plastid genomes. Several genes have been transferred from the chromatophore genome to the host nuclear genome through endosymbiotic gene transfer (EGT). Some EGT-derived proteins could be imported into chromatophores for function. Two photosynthesis-related genes (psaI and csos4A) are encoded by both the nuclear and chromatophore genomes, suggesting that EGT in Paulinella chromatophora is ongoing. Many EGT-derived genes encode proteins that function in photosynthesis and photoprotection, including an expanded family of high-light-inducible (ncHLI) proteins. Cyanobacterial hli genes are high-light induced and required for cell viability under excess light. We examined the impact of light on Paulinella chromatophora and found that this organism is light sensitive and lacks light-induced transcriptional regulation of chromatophore genes and most EGT-derived nuclear genes. However, several ncHLI genes have reestablished light-dependent regulation, which appears analogous to what is observed in cyanobacteria. We postulate that expansion of the ncHLI gene family and its regulation may reflect the light/oxidative stress experienced by Paulinella chromatophora as a consequence of the as yet incomplete integration of host and chromatophore metabolisms.

  9. Expression and immunolocalization of membrane progesterone receptors in the bovine oviduct.

    PubMed

    Kowalik, M K; Martyniak, M; Rekawiecki, R; Kotwica, J

    2016-04-01

    The oviduct plays a crucial role in the transport and maturation of gametes and ensures suitable conditions for fertility and early embryo development. One regulator of oviduct function is progesterone (P4), which affects the cell by interacting with nuclear progesterone receptors (PGRs) and through nongenomic mechanisms, presumably involving membrane PGRs. The aim of this study was to evaluate the expression of messenger RNAS (mRNAs) and proteins for progesterone receptor membrane component (PGRMC) 1 and 2 and membrane progestin receptors (mPR) α, β, and γ and to use immunohistochemistry to demonstrate their cell-specific localization in the bovine oviduct. Oviducts ipsilateral and contralateral to the corpus luteum or to the dominant follicle were collected from cows on days 6 to 12 (midluteal stage) and 18 to 20 (follicular stage) of the estrous cycle and divided into 3 parts (infundibulum, ampulla, and isthmus). There were no differences (P > 0.05) in the PGRMC1, PGRMC2, mPRα, β, and γ mRNA expression between ipsi- and contralateral oviducts. However, the same parts of the oviduct collected during the different stages of the estrous cycle showed higher (P < 0.05) mRNA levels of PGRMC1, PGRMC2, and mPRα on days 18 to 20 than on days 6 to 12 of the estrous cycle. mPRα and mPRβ mRNA levels were higher (P < 0.05) in the infundibulum than in the isthmus, whereas PGRMC1 expression was higher (P < 0.05) in the infundibulum than in ampulla. Immunohistochemistry was used to detect PGRMC1, PGRMC2, PRα, β, and γ proteins in all parts of both oviducts from days 6 to 12 and 18 to 20 of the estrous cycle. There were no differences in the staining intensity and cellular localization of the studied proteins between the ipsi- and contralateral oviducts and between the studied stages of the estrous cycle. A strong positive reaction was observed in luminal cells, but this reaction was less evident in myocytes and stromal cells. All proteins were also localized to the

  10. 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.

  11. Teicoplanin-resistant Staphylococcus aureus expresses a novel membrane protein and increases expression of penicillin-binding protein 2 complex.

    PubMed Central

    Shlaes, D M; Shlaes, J H; Vincent, S; Etter, L; Fey, P D; Goering, R V

    1993-01-01

    In the recent clinical trials of teicoplanin therapy of endocarditis caused by Staphylococcus aureus, at least one instance of the emergence of teicoplanin-resistant strains during therapy has been reported (G.W. Kaatz, S. M. Seo, N. J. Dorman, and S. A. Lerner, J. Infect. Dis 162:103-108, 1990). We have confirmed, using conventional electrophoresis of EcoRI-digested chromosomal DNA and pulsed-field gel electrophoresis of SmaI-digested chromosomal DNA, that the resistant strain (12873) (MIC, 16 micrograms/ml) is genetically very similar to the susceptible parent (12871) (MIC, 4 micrograms/ml). Kaatz et al. were able to select spontaneous teicoplanin-resistant mutants (10(-9)), suggesting that a single gene might be involved. We have shown that the mutation is highly stable during growth in the absence of teicoplanin. Using Tn551, we have selected insertion mutants of 12873 that become teicoplanin susceptible. We have examined a number of aspects of cell wall physiology in strains 12871 and 12873 and the teicoplanin-susceptible Tn551 mutants of 12873. 12873 was more susceptible to lysostaphin lysis than 12871 and the susceptible Tn551 derivatives of 12873. Autolysis in phosphate buffer (pH 7.5) and cell wall turnover rates were similar in 12871 and 12873. An analysis of membrane proteins revealed the expression of a ca. 35-kDa protein and increased expression of both polypeptides of penicillin-binding protein (PBP) 2 (PBP2) in 12873 relative to 12871 and the Tn551 mutants of 12873. This increased expression was not related to PBP2', since both strains were susceptible to oxacillin in 2% NaCl (MIC, < or = 0.25 microgram/ml) and cellular DNA from neither strain hybridized with a specific mec gene probe. Two independent Tn551 inserts have been mapped to a ca. 117-kb SmaI fragment of the chromosome. These data suggest the possibility that the mutation resulting in resistance to teicoplanin involves the regulation of expression of both polypeptides of PBP2 and a 35-k

  12. Differential expression of microRNAs with progression of gestation and inflammation in the human chorioamniotic membranes

    PubMed Central

    Montenegro, Daniel; Romero, Roberto; Pineles, Beth L.; Tarca, Adi L.; Kim, Yeon Mee; Draghici, Sorin; Kusanovic, Juan Pedro; Kim, Jung-Sun; Erez, Offer; Mazaki-Tovi, Shali; Hassan, Sonia; Espinoza, Jimmy; Kim, Chong Jai

    2010-01-01

    Objective The aim of this study was to identify differential expression of microRNAs (miRNAs) in chorioamniotic membranes with advancing gestation, labor, and inflammation. Study design Expression profiles of 157 miRNAs in the chorioamniotic membranes were obtained from patients in the following groups: 1) term not in labor (n=10); 2) term in labor (n=10); 3) preterm labor with histologic chorioamnionitis (n=9); and 4) without histologic chorioamnionitis (n=10). Results More than 95% of the miRNAs screened were expressed. Gestational age-dependent changes in expression were observed for 13 miRNAs. No differences in miRNA expression were observed between women without labor and women in labor. Membranes with chorioamnionitis displayed increased expression of miR-223 and miR-338. Gene Ontology analysis of genes targeted by differentially expressed miRNAs revealed enrichment for specific biological process categories. Conclusion Chorioamniotic membranes with advancing gestational age and chorioamnionitis are associated with the differential expression of a subset of miRNAs. PMID:17826424

  13. In silico studies of outer membrane of Neisseria meningitidis por a: its expression and immunogenic properties.

    PubMed

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Irani, Shiva

    2014-01-01

    Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis.

  14. In Silico Studies of Outer Membrane of Neisseria Meningitidis Por A: Its Expression and Immunogenic Properties

    PubMed Central

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Irani, Shiva

    2014-01-01

    Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis. PMID:25317403

  15. 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.

  16. Estrogens Induce Expression of Membrane-Associated Estrogen Receptor α Isoforms in Lactotropes

    PubMed Central

    Zárate, Sandra; Jaita, Gabriela; Ferraris, Jimena; Eijo, Guadalupe; Magri, María L.; Pisera, Daniel; Seilicovich, Adriana

    2012-01-01

    Estrogens are key to anterior pituitary function, stimulating hormone release and controlling cell fate to achieve pituitary dynamic adaptation to changing physiological conditions. In addition to their classical mechanism of action through intracellular estrogen receptors (ERs), estrogens exert rapid actions via cell membrane-localized ERs (mERs). We previously showed that E2 exerts a rapid pro-apoptotic action in anterior pituitary cells, especially in lactotropes and somatotropes, through activation of mERs. In the present study, we examined the involvement of mERα in the rapid pro-apoptotic action of estradiol by TUNEL in primary cultures of anterior pituitary cells from ovariectomized rats using a cell-impermeable E2 conjugate (E2-BSA) and an ERα selective antagonist (MPP dihydrochloride). We studied mERα expression during the estrous cycle and its regulation by gonadal steroids in vivo by flow cytometry. We identified ERα variants in the plasma membrane of anterior pituitary cells during the estrous cycle and studied E2 regulation of these mERα variants in vitro by surface biotinylation and Western Blot. E2-BSA-induced apoptosis was abrogated by MPP in total anterior pituitary cells and lactotropes. In cycling rats, we detected a higher number of lactotropes and a lower number of somatotropes expressing mERα at proestrus than at diestrus. Acute E2 treatment increased the percentage of mERα-expressing lactotropes whereas it decreased the percentage of mERα-expressing somatotropes. We detected three mERα isoforms of 66, 39 and 22 kDa. Expression of mERα66 and mERα39 was higher at proestrus than at diestrus, and short-term E2 incubation increased expression of these two mERα variants. Our results indicate that the rapid apoptotic action exerted by E2 in lactotropes depends on mERα, probably full-length ERα and/or a 39 kDa ERα variant. Expression and activation of mERα variants in lactotropes could be one of the mechanisms through which E2

  17. Integrated profiling of microRNA expression in membranous nephropathy using high-throughput sequencing technology.

    PubMed

    Chen, Wenbiao; Lin, Xiaocong; Huang, Jianrong; Tan, Kuibi; Chen, Yuyu; Peng, Wujian; Li, Wuxian; Dai, Yong

    2014-01-01

    The present study analyzed microRNA (miRNA) expression profiles in peripheral blood lymphocyte cells (PBLCs) from patients with membranous nephropathy (MN) and normal controls (NC), in an effort to improve the understanding of the pathogenesis of MN. High-throughput sequencing was performed on 30 MN patients and 30 healthy individuals (NC group). Known and novel miRNAs were analyzed and the results were confirmed by quantitative reverse transcription PCR (qRT-PCR). In total, 326 miRNAs showed a significant difference in expression between the MN and NC groups. This included 286 downregulated miRNAs and 40 upregulated miRNAs. In addition, there were 6 novel miRNAs that presented differential levels of expression between the MN and NC groups. The miRNAs were mapped to the genome, using a short oligonucleotide alignment program (SOAP), to analyze their expression and distribution. Twenty-five percent of the unique miRNAs in the MN group and 52.1% in the NC group were mapped to the genome. One hundred and eight mismatches were identified. Seventy-seven mismatches were detected in a higher proportion of the MN samples, compared with the NC samples. Twenty-five mismatches were detected in a higher proportion of the NC samples than the MN samples. Differential miRNA expression was also detected between 10 randomly selected pair groups, as depicted in a cluster analysis diagram. These data indicate that differential miRNA expression may be involved in the pathogenesis of MN. In addition, the discrepancies between the MN and NC groups, in the mismatched miRNAs that were mapped to the genome, strongly suggest that miRNAs play an important role in the pathogenesis of human disorders. miRNAs may provide a potential breakthrough in the research of MN and may provide a novel biomarker for the diagnosis and treatment of the disease.

  18. Development of the photosynthetic apparatus of Cunninghamia lanceolata in light and darkness.

    PubMed

    Xue, Xian; Wang, Qi; Qu, Yanli; Wu, Hongyang; Dong, Fengqin; Cao, Haoyan; Wang, Hou-Ling; Xiao, Jianwei; Shen, Yingbai; Wan, Yinglang

    2017-01-01

    Here, we compared the development of dark- and light-grown Chinese fir (Cunninghamia lanceolata) cotyledons, which synthesize chlorophyll in the dark, representing a different phenomenon from angiosperm model plants. We determined that the grana lamellar membranes were well developed in both chloroplasts and etiochloroplasts. The accumulation of thylakoid membrane protein complexes was similar between chloroplasts and etiochloroplasts. Measurement of chlorophyll fluorescence parameters indicated that photosystem II (PSII) had low photosynthetic activities, whereas the photosystem I (PSI)-driven cyclic electron flow (CEF) rate exceeded the rate of PSII-mediated photon harvesting in etiochloroplasts. Analysis of the protein contents in etiochloroplasts indicated that the light-harvesting complex II remained mostly in its monomeric conformation. The ferredoxin NADP(+) oxidoreductase and NADH dehydrogenase-like complexes were relatively abundantly expressed in etiochloroplasts for Chinese fir. Our transcriptome analysis contributes a global expression database for Chinese fir cotyledons, providing background information on the regulatory mechanisms of different genes involved in the development of dark- and light-grown cotyledons. In conclusion, we provide a novel description of the early developmental status of the light-dependent and light-independent photosynthetic apparatuses in gymnosperms.

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

    PubMed

    Ji, Peng; Lodish, Harvey F

    2012-01-27

    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.

  20. Evolving a photosynthetic organelle.

    PubMed

    Nakayama, Takuro; Archibald, John M

    2012-04-24

    The evolution of plastids from cyanobacteria is believed to represent a singularity in the history of life. The enigmatic amoeba Paulinella and its 'recently' acquired photosynthetic inclusions provide a fascinating system through which to gain fresh insight into how endosymbionts become organelles.The plastids, or chloroplasts, of algae and plants evolved from cyanobacteria by endosymbiosis. This landmark event conferred on eukaryotes the benefits of photosynthesis--the conversion of solar energy into chemical energy--and in so doing had a huge impact on the course of evolution and the climate of Earth 1. From the present state of plastids, however, it is difficult to trace the evolutionary steps involved in this momentous development, because all modern-day plastids have fully integrated into their hosts. Paulinella chromatophora is a unicellular eukaryote that bears photosynthetic entities called chromatophores that are derived from cyanobacteria and has thus received much attention as a possible example of an organism in the early stages of organellogenesis. Recent studies have unlocked the genomic secrets of its chromatophore 23 and provided concrete evidence that the Paulinella chromatophore is a bona fide photosynthetic organelle 4. The question is how Paulinella can help us to understand the process by which an endosymbiont is converted into an organelle.

  1. 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

  2. Nerve growth cones isolated from fetal rat brain. IV. Preparation of a membrane subfraction and identification of a membrane glycoprotein expressed on sprouting neurons

    PubMed Central

    1985-01-01

    This study describes the preparation of a membrane subfraction from isolated nerve growth cone particles (GCPs) (see Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Friedman, and S. Somlo, 1983, Cell, 35:573- 584) and the identification in this fraction of a glycoprotein expressed during neurite growth. While approximately 40 major polypeptides are visible in Coomassie Blue-stained SDS polyacrylamide gels of pelleted (partially disrupted) GCPs, a salt-washed membrane fraction prepared from lysed, detergent-permeabilized GCPs contains only 14% of this protein and has an unusually simple polypeptide pattern of seven major bands. Monoclonal antibodies have been generated to GCP membranes isolated from fetal rat brain. These antibodies have been screened differentially with synaptosomes from adult rat brain in order to identify those which recognize antigens expressed selectively during neurite growth. One such antibody (termed 5B4) recognizes a developmentally regulated membrane glycoprotein that is enriched in GCP membranes and expressed in fetal neurons sprouting in vitro. The 5B4 antigen in fetal brain migrates in SDS polyacrylamide gels as a diffuse band of approximately 185-255 kD, is rich in sialic acid, and consists of a small family of isoelectric variants. Freezing-thawing and neuraminidase digestion result in the cleavage of the native antigen into two new species migrating diffusely around 200 and 160 kD. Prolonged neuraminidase digestion sharpens these bands at about 180 and 135 kD, respectively. In the mature brain, antibody 5B4 recognizes a sparse polypeptide migrating at approximately 140 kD. As shown in the following paper (Wallis, I., L. Ellis, K. Suh, and K. H. Pfenninger, 1985, J. Cell Biol., 101:1990-1998), the fetal antigen is specifically associated with regions of neuronal sprouting and, therefore, can be used as a molecular marker of neurite growth. PMID:3902858

  3. 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.

  4. 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

  5. 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

    2015-10-05

    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.

  6. 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

  7. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas.

    PubMed

    Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G; Kristensen, Bjarne

    2013-01-01

    Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm.

  8. 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.

  9. Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

    PubMed

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-05-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

  10. Chorioamnionitis and increased galectin-1 expression in PPROM –an anti-inflammatory response in the fetal membranes?

    PubMed Central

    Than, Nandor Gabor; Kim, Sung-Su; Abbas, Asad; Han, Yu Mi; Hotra, John; Tarca, Adi L.; Erez, Offer; Wildman, Derek E.; Kusanovic, Juan Pedro; Pineles, Beth; Montenegro, Daniel; Edwin, Samuel S.; Mazaki-Tovi, Shali; Gotsch, Francesca; Espinoza, Jimmy; Hassan, Sonia S.; Papp, Zoltan; Romero, Roberto

    2008-01-01

    Problem Galectin-1 can regulate immune responses upon infection and inflammation. We determined galectin-1 expression in the chorioamniotic membranes and its changes during histological chorioamnionitis. Methods of Study Chorioamniotic membranes were obtained from women with normal pregnancy (n=5) and from patients with pre-term pre-labor rupture of the membranes (PPROM) with (n=8) and without histological chorioamnionitis (n=8). Galectin-1 mRNA and protein were localized by in situ hybridization and immunohistochemistry. Galectin-1 mRNA expression was also determined by quantitative RT-PCR. Results Galectin-1 mRNA and protein were detected in the amnion epithelium, chorioamniotic fibroblasts/myofibroblasts and macrophages, chorionic trophoblasts, and decidual stromal cells. In patients with PPROM, galectin-1 mRNA expression in the fetal membranes was higher (2.07-fold, p=0.002) in those with chorioamnionitis than in those without. Moreover, chorioamionitis was associated with a strong galectin-1 immunostaining in amniotic epithelium, chorioamniotic mesodermal cells, and apoptotic bodies. Conclusions Chorioamnionitis is associated with an increased galectin-1 mRNA expression and strong immunoreactivity of the chorioamniotic membranes; thus, galectin-1 may be involved in the regulation of the inflammatory responses to chorioamniotic infection. PMID:18691335

  11. Studies on the expression of outer membrane protein 2 in escherichia coli.

    PubMed

    Fralick, J A; Diedrich, D L

    1982-01-01

    The relative level of protein 2 expressed in the outer membrane of strains of Escherichia coli K-12 lysogenized with bacteriophage PA-2 was found to be influenced by both the growth temperature and lc+ gene dosage. An increase in either of these parameters was accompanied by an increase in the level of protein 2 up to an apparent saturation level. Any increase in the amount of protein 2 was accompanied by a concomittant decrease in the amount of OmpF and OmpC porins. This inverse relationship led to the maintenance of an approximately constant protein mass per unit of peptidoglycan. Our results are discussed in light of recent genetic studies on the regulation of the OmpF and OmpC porins and can be explained through the competition of these three matrix proteins for a common export or insertion site.

  12. Brassica napus responses to short-term excessive copper treatment with decrease of photosynthetic pigments, differential expression of heavy metal homeostasis genes including activation of gene NRAMP4 involved in photosystem II stabilization.

    PubMed

    Zlobin, I E; Kholodova, V P; Rakhmankulova, Z F; Kuznetsov, Vl V

    2015-08-01

    In the present study, the influence of 50 and 100 µM CuSO4 was investigated starting from 3 h till 72 h treatment of 4-weeks Brassica napus plants. High CuSO4 concentrations in nutrient medium resulted in the rapid copper accumulation in plants, especially in roots, much slower and to lower degree in leaves. Copper excess induced early decrease in the leaf water content and temporary leaf wilting. The decrease in content of photosynthetic pigments became significant to 24 h of excessive copper treatments and reached 35 % decrease to 72 h, but there were no significant changes in maximum quantum efficiency of photosystem II photochemistry. The copper excess affected the expression of ten genes involved in heavy metal homeostasis and copper detoxification. The results showed the differential and organ-specific expression of most genes. The potential roles of copper-activated genes encoding heavy metal transporters (ZIP5, NRAMP4, YSL2, and MRP1), metallothioneins (MT1a and MT2b), low-molecular chelator synthesis enzymes (PCS1 and NAS2), and metallochaperones (CCS and HIPP06) in heavy metal homeostasis and copper ion detoxification were discussed. The highest increase in gene expression was shown for NRAMP4 in leaves in spite of relatively moderate Cu accumulation there. The opinion was advanced that the NRAMP4 activation can be considered among the early reactions in the defense of the photosystem II against copper excess.

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

    PubMed

    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.

  14. Membrane biogenesis during B cell differentiation: most endoplasmic reticulum proteins are expressed coordinately

    PubMed Central

    1990-01-01

    The induction of high-rate protein secretion entails increased biogenesis of secretory apparatus organelles. We examined the biogenesis of the secretory apparatus in the B cell line CH12 because it can be induced in vitro to secrete immunoglobulin (Ig). Upon stimulation with lipopolysaccharide (LPS), CH12 cells increased secretion of IgM 12-fold. This induced secretion was accompanied by preferential expansion of the ER and the Golgi complex. Three parameters of the rough ER changed: its area and volume increased 3.3- and 3.7-fold, respectively, and the density of membrane-bound ribosomes increased 3.5-fold. Similarly, the area of the Golgi stack increased 3.3-fold, and its volume increased 4.1-fold. These changes provide sufficient biosynthetic capacity to account for the increased secretory activity of CH12. Despite the large increase in IgM synthesis, and because of the expansion of the ER, the concentration of IgM within the ER changed less than twofold during the differentiation process. During the amplification of the rough ER, the expression of resident proteins changed according to one of two patterns. The majority (75%) of rough microsomal (RM) proteins increased in proportion to the increase in rough ER size. Included in this group were both lumenal proteins such as Ig binding protein (BiP), and membrane proteins such as ribophorins I and II. In addition, the expression of a minority (approximately 9%) of RM polypeptides increased preferentially, such that their abundance within the RM of secreting CH12 cells was increased. Thus, the expansion of ER during CH12 differentiation involves preferential increases in the abundance of a few resident proteins, superimposed upon proportional increases in most ER proteins. PMID:2335560

  15. Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.

    PubMed Central

    Bartley, G E; Viitanen, P V; Pecker, I; Chamovitz, D; Hirschberg, J; Scolnik, P A

    1991-01-01

    Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins. Images PMID:1862081

  16. Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum, which possesses two complete sets of the genes.

    PubMed Central

    Viale, A M; Kobayashi, H; Akazawa, T

    1989-01-01

    Two sets of genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were detected in the photosynthetic purple sulfur bacterium Chromatium vinosum by hybridization analysis with RuBisCO gene probes, cloned by using the lambda Fix vector, and designated rbcL-rbcS and rbcA-rbcB. rbcL and rbcA encode the large subunits, and rbcS and rbcB encode the small subunits. rbcL-rbcS was the same as that reported previously (A. M. Viale, H. Kobayashi, T. Takabe, and T. Akazawa, FEBS Lett. 192:283-288, 1985). A DNA fragment bearing rbcA-rbcB was subcloned in plasmid vectors and sequenced. We found that rbcB was located 177 base pairs downstream of the rbcA coding region, and both genes were preceded by plausible procaryotic ribosome-binding sites. rbcA and rbcD encoded polypeptides of 472 and 118 amino acids, respectively. Edman degradation analysis of the subunits of RuBisCO isolated from C. vinosum showed that rbcA-rbcB encoded the enzyme present in this bacterium. The large- and small-subunit polypeptides were posttranslationally processed to remove 2 and 1 amino acid residues from their N-termini, respectively. Among hetero-oligomeric RuBisCOs, the C. vinosum large subunit exhibited higher homology to that from cyanobacteria, eucaryotic algae, and higher plants (71.6 to 74.2%) than to that from the chemolithotrophic bacterium Alcaligenes eutrophus (56.6%). A similar situation has been observed for the C. vinosum small subunit, although the homology among small subunits from different organisms was lower than that among the large subunits. Images PMID:2708310

  17. Photosynthetic Photovoltaic Cells

    DTIC Science & Technology

    2007-06-21

    PHOTOSYNTHETIC PHOTOVOLTAIC CELLS 5b. GRANT NUMBER F49620-02-1-0399 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER MARC A. BALDO 5e. TASK...building an ’antenna’ on top of a conventional solar cell. Biomimetic organic solar cells operate as follows: The antenna absorbs the light, and acts to...no longer must absorb all the light. Thus, its quantum efficiency can approach 100% potentially doubling the performance of organic solar cells. 15

  18. Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana.

    PubMed

    Strand, A; Zrenner, R; Trevanion, S; Stitt, M; Gustafsson, P; Gardeström, P

    2000-09-01

    Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi-limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose-1,5-bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose-P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end-product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP-glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards

  19. Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia coli.

    PubMed Central

    Duchêne, M; Barron, C; Schweizer, A; von Specht, B U; Domdey, H

    1989-01-01

    Lipoprotein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. Like porin protein F (OprF), it is a vaccine candidate because it antigenically cross-reacts with all serotype strains of the International Antigenic Typing Scheme. Since lipoprotein I was expressed in Escherichia coli under the control of its own promoter, we were able to isolate the gene by screening a lambda EMBL3 phage library with a mouse monoclonal antibody directed against lipoprotein I. The monocistronic OprI mRNA encodes a precursor protein of 83 amino acid residues including a signal peptide of 19 residues. The mature protein has a molecular weight of 6,950, not including bound glycerol and lipid. Although the amino acid sequences of protein I of P. aeruginosa and Braun's lipoprotein of E. coli differ considerably (only 30.1% identical amino acid residues), peptidoglycan in E. coli, are identical. Using lipoprotein I expressed in E. coli, it can now be tested whether this protein alone, without P. aeruginosa lipopolysaccharide contaminations, has a protective effect against P. aeruginosa infections. Images PMID:2502533

  20. Mechanism of GABAB receptor-induced BDNF secretion and promotion of GABAA receptor membrane expression.

    PubMed

    Kuczewski, Nicola; Fuchs, Celine; Ferrand, Nadine; Jovanovic, Jasmina N; Gaiarsa, Jean-Luc; Porcher, Christophe

    2011-08-01

    Recent studies have shown that GABA(B) receptors play more than a classical inhibitory role and can function as an important synaptic maturation signal early in life. In a previous study, we reported that GABA(B) receptor activation triggers secretion of brain-derived neurotrophic factor (BDNF) and promotes the functional maturation of GABAergic synapses in the developing rat hippocampus. To identify the signalling pathway linking GABA(B) receptor activation to BDNF secretion in these cells, we have now used the phosphorylated form of the cAMP response element-binding protein as a biological sensor for endogenous BDNF release. In the present study, we show that GABA(B) receptor-induced secretion of BDNF relies on the activation of phospholipase C, followed by the formation of diacylglycerol, activation of protein kinase C, and the opening of L-type voltage-dependent Ca(2+) channels. We further show that once released by GABA(B) receptor activation, BDNF increases the membrane expression of β(2/3) -containing GABA(A) receptors in neuronal cultures. These results reveal a novel function of GABA(B) receptors in regulating the expression of GABA(A) receptor through BDNF-tropomyosin-related kinase B receptor dependent signalling pathway.

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

    PubMed Central

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

    2015-01-01

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

  2. Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from Thermus thermophilus HB27

    PubMed Central

    Brosig, Alexander; Nesper, Jutta; Welte, Wolfram; Diederichs, Kay

    2008-01-01

    The cell envelope of the thermophilic bacterium Thermus thermophilus is multilayered and includes an outer membrane with integral outer membrane proteins that are not well characterized. The hypothetical protein TTC0834 from T. thermophilus HB27 was identified as a 22 kDa outer membrane protein containing eight predicted β-strands. TTC0834 was expressed with an N-­terminal His tag in T. thermophilus HB8 and detected in the S-layer/outer membrane envelope fraction. His-TTC0834 was purified and crystallized under various conditions. Native data sets were collected to 3.2 Å resolution and the best diffracting crystals belonged to space group P3121 or P3221, with unit-cell parameters a = b = 166.67, c = 97.53 Å. PMID:18540069

  3. Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from Thermus thermophilus HB27.

    PubMed

    Brosig, Alexander; Nesper, Jutta; Welte, Wolfram; Diederichs, Kay

    2008-06-01

    The cell envelope of the thermophilic bacterium Thermus thermophilus is multilayered and includes an outer membrane with integral outer membrane proteins that are not well characterized. The hypothetical protein TTC0834 from T. thermophilus HB27 was identified as a 22 kDa outer membrane protein containing eight predicted beta-strands. TTC0834 was expressed with an N-terminal His tag in T. thermophilus HB8 and detected in the S-layer/outer membrane envelope fraction. His-TTC0834 was purified and crystallized under various conditions. Native data sets were collected to 3.2 A resolution and the best diffracting crystals belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 166.67, c = 97.53 A.

  4. 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.

  5. 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

  6. Photosynthetic Pigments in Diatoms.

    PubMed

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

    2015-09-16

    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.

  7. WY14,643, a PPARα ligand, attenuates expression of anti-glomerular basement membrane disease

    PubMed Central

    Archer, D C; Frkanec, J T; Cromwell, J; Clopton, P; Cunard, R

    2007-01-01

    Peroxisome proliferator-activated receptor alpha (PPARα) ligands are medications used to treat hyperlipidaemia and atherosclerosis. Increasing evidence suggests that these agents are immunosuppressive. In the following studies we demonstrate that WY14,643, a PPARα ligand, attenuates expression of anti-glomerular basement membrane disease (AGBMD). C57BL/6 mice were fed 0·05% WY14,643 or control food and immunized with the non-collagenous domain of the α3 chain of Type IV collagen [α3(IV) NC1] in complete Freund's adjuvant (CFA). WY14,643 reduced proteinuria and greatly improved glomerular and tubulo-interstitial lesions. However, the PPARα ligand did not alter the extent of IgG-binding to the GBM. Immunohistochemical studies revealed that the prominent tubulo-interstitial infiltrates in the control-fed mice consisted predominately of F4/80+ macrophages and WY14,643-feeding decreased significantly the number of renal macrophages. The synthetic PPARα ligand also reduced significantly expression of the chemokine, monocyte chemoattractant protein (MCP)-1/CCL2. Sera from mice immunized with AGBMD were also evaluated for antigen-specific IgGs. There was a significant increase in the IgG1 : IgG2c ratio and a decline in the intrarenal and splenocyte interferon (IFN)-γ mRNA expression in the WY14,643-fed mice, suggesting that the PPARα ligand could skew the immune response to a less inflammatory T helper 2-type of response. These studies suggest that PPARα ligands may be a novel treatment for inflammatory renal disease. PMID:17888025

  8. 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

  9. PTK6/BRK is expressed in the normal mammary gland and activated at the plasma membrane in breast tumors.

    PubMed

    Peng, Maoyu; Emmadi, Rajyasree; Wang, Zebin; Wiley, Elizabeth L; Gann, Peter H; Khan, Seema A; Banerji, Nilanjana; McDonald, William; Asztalos, Szilard; Pham, Thao N D; Tonetti, Debra A; Tyner, Angela L

    2014-08-15

    Protein Tyrosine kinase 6 (PTK6/BRK) is overexpressed in the majority of human breast tumors and breast tumor cell lines. It is also expressed in normal epithelial linings of the gastrointestinal tract, skin, and prostate. To date, expression of PTK6 has not been extensively examined in the normal human mammary gland. We detected PTK6 mRNA and protein expression in the immortalized normal MCF-10A human mammary gland epithelial cell line, and examined PTK6 expression and activation in a normal human breast tissue microarray, as well as in human breast tumors. Phosphorylation of tyrosine residue 342 in the PTK6 activation loop corresponds with its activation. Similar to findings in the prostate, we detect nuclear and cytoplasmic PTK6 in normal mammary gland epithelial cells, but no phosphorylation of tyrosine residue 342. However, in human breast tumors, striking PTK6 expression and phosphorylation of tyrosine 342 is observed at the plasma membrane. PTK6 is expressed in the normal human mammary gland, but does not appear to be active and may have kinase-independent functions that are distinct from its cancer promoting activities at the membrane. Understanding consequences of PTK6 activation at the plasma membrane may have implications for developing novel targeted therapies against this kinase.

  10. Statin therapy and the expression of genes that regulate calcium homeostasis and membrane repair in skeletal muscle.

    PubMed

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

    2010-07-01

    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.

  11. Nuclear and membrane progestin receptors in the European eel: Characterization and expression in vivo through spermatogenesis.

    PubMed

    Morini, Marina; Peñaranda, David S; Vílchez, María C; Nourizadeh-Lillabadi, Rasoul; Lafont, Anne-Gaëlle; Dufour, Sylvie; Asturiano, Juan F; Weltzien, Finn-Arne; Pérez, Luz

    2017-02-11

    Characterization of all the progestin receptor genes (PRs) found in the European eel has been performed. There were five membrane PRs (mPRs): mPRα (alpha), mPRAL1 (alpha-like1), mPRAL2 (alpha-like2), mPRγ (gamma), mPRδ (delta) and two nuclear PRs (nPRs or PGRs): pgr1 and pgr2. In silico studies showed that the C and E(F) domains of Pgr are well conserved among vertebrates whereas the A/B domain is not. Phylogeny and synteny analyses suggest that eel duplicated pgr (pgr1 and pgr2) originated from the teleost-specific third whole genome duplication (3R). mPR phylogeny placed three eel mPRs together with the mPRα clade, being termed mPRα, mPRAL1 and mPRAL2, while the other two eel mPRs clustered with mPRγ and mPRδ clades, respectively. The in vivo study showed differential expression patterns along the brain-pituitary-gonad axis. An increase in nPR transcripts was observed in brain (in pgr1) and pituitary (in pgr1 and pgr2) through the spermatogenesis, from the spermatogonia B/spermatocyte stage to the spermiation stage. In the testis, mPRγ, mPRδ and pgr2 transcripts showed the highest levels in testis with A spermatogonia as dominant germ cell, while the highest mPRα, mPRAL1 and mPRAL2 transcripts were observed in testis from spermiating males, where the dominant germ cell were spermatozoa. Further studies should elucidate the role of both nuclear and membrane progestin receptors on eel spermatogenesis.

  12. 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.

  13. Construction and expression of bivalent membrane-anchored DNA vaccine encoding Sjl4FABP and Sj26GST genes.

    PubMed

    Guo, Ping; Dai, Wuxing; Liu, Shuojie; Yang, Ping; Cheng, Jizhong; Liang, Liang; Chen, Zhihao; Gao, Hong

    2006-01-01

    In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjcl4FABP and Sjc26GST genes and identify their expression in vitro, Sj14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 (IL-2) upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human placental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES, resulting in another new recombinant plasmid pIRES-Sj26-Sj14. The expression of Sj14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj14 were successfully constructed and the expression of modified Sj14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.

  14. 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.

  15. Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery.

    PubMed

    Panavas, Tadas; Lu, Jin; Liu, Xuesong; Winkis, Ann-Marie; Powers, Gordon; Naso, Michael F; Amegadzie, Bernard

    2011-09-01

    Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.

  16. 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

  17. Cytochrome c oxidase is regulated by modulations in protein expression and mitochondrial membrane phospholipid composition in estivating African lungfish.

    PubMed

    Frick, N T; Bystriansky, J S; Ip, Y K; Chew, S F; Ballantyne, J S

    2010-03-01

    We examined some of the potential mechanisms lungfish (Protopterus dolloi) use to regulate cytochrome c oxidase (CCO), during metabolic depression. CCO activity was reduced by 67% in isolated liver mitochondria of estivating fish. This was likely accomplished, in part, by the 46% reduction in CCO subunit I protein expression in the liver. No change in the mRNA expression levels of CCO subunits I, II, III, and IV were found in the liver, suggesting CCO is under translational regulation; however, in the kidney, messenger limitation may be a factor as the expression of subunits I and II were depressed ( approximately 10-fold) during estivation, suggesting tissue-specific mechanisms of regulation. CCO is influenced by mitochondrial membrane phospholipids, particularly cardiolipin (CL). In P. dolloi, the phospholipid composition of the liver mitochondrial membrane changed during estivation, with a approximately 2.3-fold reduction in the amount of CL. Significant positive correlations were found between CCO activity and the amount of CL and phosphatidylethanolamine within the mitochondrial membrane. It appears CCO activity is regulated through multiple mechanisms in P. dolloi, and individual subunits of CCO are regulated independently, and in a tissue-specific manner. It is proposed that altering the amount of CL within the mitochondrial membrane may be a means of regulating CCO activity during metabolical depression in the African lungfish, P. dolloi.

  18. Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38).

    PubMed Central

    Luzio, J P; Brake, B; Banting, G; Howell, K E; Braghetta, P; Stanley, K K

    1990-01-01

    Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network. Images Fig. 1. Fig. 3. PMID:2204342

  19. Recombinant Phospholipase A1 of the Outer Membrane of Psychrotrophic Yersinia pseudotuberculosis: Expression, Purification, and Characterization.

    PubMed

    Bakholdina, S I; Tischenko, N M; Sidorin, E V; Isaeva, M P; Likhatskaya, G N; Dmitrenok, P S; Kim, N Yu; Chernikov, O V; Solov'eva, T F

    2016-01-01

    The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.

  20. Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system.

    PubMed

    Lewinson, D; Maor, G; Rozen, N; Rabinovich, I; Stahl, S; Rachmiel, A

    2001-11-01

    An in vivo system of membranous bone formation during distraction has been investigated in order to follow cells that express vascular markers with the objective of understanding the neovascularization process. Concomitantly, sustained proliferation of preskeletal cells was achieved through the application of mechanical force. New capillaries and leading edges that arose by angiogenesis from the periosteal and mucosal surfaces and invaded the central zone of the regenerating distraction tissue temporally preceded the growth of delicate woven bone trabeculae from both edges of the cut bone. Concentrically arranged 'onion-like' configurations were abundant in paracentral zones and in association with mesenchymal condensations, suggesting their de novo formation in situ. Vascular specific markers, the angiopoietin receptor Tie-2 and factor VIII-related antigen (FVIIIrAg), were localized immunohistochemically in order to follow cells of vascular origin. Endothelial cells of the new capillaries, centrally located cells of the concentric configurations, pericytes, and most of the adjacent polygonal mesenchymal cells stained positively with specific antibodies to both antigens. Moreover, preosteoblasts and osteoblasts that lie adjacent to or already embedded in the osteiod of the newly formed trabeculae were also FVIIIrAg and Tie-2 immunopositive. As the source of the bone-forming cells in regenerating tissue during distraction is not yet fully understood, this observation might support the possibility of their vascular origin.

  1. Two plasma membrane H(+)-ATPase genes are differentially expressed in iron-deficient cucumber plants.

    PubMed

    Santi, Simonetta; Cesco, Stefano; Varanini, Zeno; Pinton, Roberto

    2005-03-01

    Aim of the present work was to investigate the involvement of plasma membrane (PM) H(+)-ATPase (E.C. 3.6.3.6) isoforms of cucumber (Cucumis sativus L.) in the response to Fe deficiency. Two PM H(+)-ATPase cDNAs (CsHA1 and CsHA2) were isolated from cucumber and their expression analysed as a function of Fe nutritional status. Semi-quantitative reverse transcriptase (RT)-PCR and quantitative real-time RT-PCR revealed in Fe-deficient roots an enhanced accumulation of CsHA1 gene transcripts, which were hardly detectable in leaves. Supply of iron to deficient plants caused a decrease in the transcript level of CsHA1. In contrast, CsHA2 transcripts, detected both in roots and leaves, appeared to be unaffected by Fe. This work shows for the first time that a transcriptional regulation of PM H(+)-ATPase involving a specific isoform occurs in the response to Fe deficiency.

  2. 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.

  3. Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling.

    PubMed

    Silver, Kristopher; Littlejohn, Alaina; Thomas, Laurel; Marsh, Elizabeth; Lillich, James D

    2015-12-15

    Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

  4. Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling

    PubMed Central

    Silver, Kristopher; Littlejohn, Alaina; Thomas, Laurel; Marsh, Elizabeth; Lillich, James D.

    2015-01-01

    Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40 mM K+, or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity. PMID:26549367

  5. 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.

  6. Photosynthetic approaches to chemical biotechnology.

    PubMed

    Desai, Shuchi H; Atsumi, Shota

    2013-12-01

    National interest and environmental advocates encourage alternatives to petroleum-based products. Besides biofuels, many other valuable chemicals used in every-day life are petroleum derivatives or require petroleum for their production. A plausible alternative to production using petroleum for chemical production is to harvest the abundant carbon dioxide resources in the environment to produce valuable hydrocarbons. Currently, efforts are being made to utilize a natural biological system, photosynthetic microorganisms, to perform this task. Photosynthetic microorganisms are attractive to use for biochemical production because they utilize economical resources for survival: sunlight and carbon dioxide. This review examines the various compounds produced by photosynthetic microorganisms.

  7. 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.

  8. Light gradients in spherical photosynthetic vesicles.

    PubMed

    Paillotin, G; Leibl, W; Gapiński, J; Breton, J; Dobek, A

    1998-07-01

    Light-gradient photovoltage measurements were performed on EDTA-treated thylakoids and on osmotically swollen thylakoids (blebs), both of spherical symmetry but of different sizes. In the case of EDTA vesicles, a negative polarity (due to the normal light gradient) was observed in the blue range of the absorption spectrum, and a positive polarity, corresponding to an inverse light gradient, was observed at lambda = 530 and lambda = 682 nm. The sign of the photovoltage polarity measured in large blebs (swollen thylakoids) is the same as that obtained for whole chloroplasts, although differences in the amplitudes are observed. An approach based on the use of polar coordinates was adapted for a theoretical description of these membrane systems of spherical symmetry. The light intensity distribution and the photovoltage in such systems were calculated. Fits to the photovoltage amplitudes, measured as a function of light wavelength, made it possible to derive the values of the dielectric constant of the protein, epsilons = 3, and the refractive index of the photosynthetic membrane for light propagating perpendicular and parallel to the membrane surface, nt = 1.42 and nn = 1.60, respectively. The latter two values determine the birefringence of the biological membrane, Deltan = nn - nt = 0.18.

  9. Photosynthetic characteristics and organization of chlorophyll in marine dinoflagellates

    PubMed Central

    Prézelin, Barbara B.; Alberte, Randall S.

    1978-01-01

    The photosystem I reaction center complex, the P-700-chlorophyll a-protein, has been isolated from the photosynthetic membranes of two marine dinoflagellates, Gonyaulax polyedra and Glenodinium sp., by detergent solubilization with Triton X-100. The complexes isolated from the two species were indistinguishable, exhibiting identical absorption properties (400-700 nm) at both room (300 K) and low (77 K) temperature. The room temperature, red wavelength maximum was at 675 nm. The absorption properties, kinetics of photobleaching, sodium dodecyl sulfate electrophoretic mobilities, and chlorophyll a/P-700 ratio (50 ± 10) of the P-700-chlorophyll a-protein complexes from the two species also were essentially the same and similar to those properties characterizing P-700-chlorophyll a-protein complexes of higher plants and green algae. Photosynthetic unit sizes were determined for cells grown at 1000 μW/cm2. Both dinoflagellates had unit sizes (total chlorophyll/P-700 ratios) of about 600, even though the distribution of chlorophyll a, chlorophyll c, and peridinin in the light-harvesting components differed in Gonyaulax and Glenodinium. The number of photosynthetic units per cell in the two species correlates directly with their photosynthetic activities. A model is presented for the distribution of chlorophyll in the photosynthetic apparatus of these dinoflagellates which accounts for the known role of the isolated pigment-protein complexes and for the known photoadaptive physiology in pigmentation and photosynthesis for these species. PMID:16592518

  10. Caveolin-1 Expression and Membrane Cholesterol Content Modulate N-Type Calcium Channel Activity in NG108-15 Cells

    PubMed Central

    Toselli, M.; Biella, G.; Taglietti, V.; Cazzaniga, E.; Parenti, M.

    2005-01-01

    Caveolins are the main structural proteins of glycolipid/cholesterol-rich plasmalemmal invaginations, termed caveolae. In addition, caveolin-1 isoform takes part in membrane remodelling as it binds and transports newly synthesized cholesterol from endoplasmic reticulum to the plasma membrane. Caveolin-1 is expressed in many cell types, including hippocampal neurons, where an abundant SNAP25-caveolin-1 complex is detected after induction of persistent synaptic potentiation. To ascertain whether caveolin-1 influences neuronal voltage-gated Ca2+ channel basal activity, we stably expressed caveolin-1 into transfected neuroblastoma × glioma NG108-15 hybrid cells [cav1(+) clone] that lack endogenous caveolins but express N-type Ca2+ channels upon cAMP-induced neuronal differentiation. Whole-cell patch-clamp recordings of cav1(+) cells demonstrated that N-type current density was reduced in size by ∼70% without any significant change in the time course of activation and inactivation and voltage dependence. Moreover, the cav1(+) clone exhibited a significantly increased proportion of membrane cholesterol compared to wild-type NG108-15 cells. To gain insight into the mechanism underlying caveolin-1 lowering of N-current density, and more precisely to test whether this was indirectly caused by caveolin-1-induced enhancement of membrane cholesterol, we compared single N-type channel activities in cav1(+) clone and wild-type NG108-15 cells enriched with cholesterol after exposure to a methyl-β-cyclodextrin-cholesterol complex. A lower Ca2+ channel activity was recorded from cell-attached patches of both cell types, thus supporting the view that the increased proportion of membrane cholesterol is ultimately responsible for the effect. This is due to a reduction in the probability of channel opening caused by a significant decrease of channel mean open time and by an increase of the frequency of null sweeps. PMID:16040758

  11. Enhanced expression and secretion of an epithelial membrane antigen (MA5) in a human mucinous breast tumor line (BT549).

    PubMed

    Williams, C J; Major, P P; Dion, A S

    1990-01-01

    The mouse monoclonal antibody MA5, generated versus a membrane-enriched extract of breast cancer metastatic to liver, detects one or two high molecular weight species (greater than 200 kD) in breast tumor membranes, human milk fat globule membranes, and various breast tumor cell lines. From comparative studies of five breast carcinoma lines (BT20, BT549, MCF-7, T47D, and ZR75-1), as well as an epithelial line established from milk (HBL-100), we report the stimulation of expression of MA5-reactive antigen in a mucinous breast tumor cell line (BT549) through the use of a culture medium supplemented with charcoal-absorbed fetal calf serum, insulin, and hydrocortisone. Large amounts of aggregated MA5-reactive antigen are secreted into the culture medium and can be recovered from the media for further purification by centrifugation. These findings suggest that BT549 cells, grown in the special nutritive medium, may be useful in providing an ample source of epithelial membrane antigen (also termed polymorphic epithelial mucin) for standardization of clinical assay protocols, as well as provide a model system for studies of the regulation of expression for this class of antigens in breast carcinoma.

  12. Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes

    PubMed Central

    Schrottke, Stefanie; Kaiser, Anette; Vortmeier, Gerrit; Els-Heindl, Sylvia; Worm, Dennis; Bosse, Mathias; Schmidt, Peter; Scheidt, Holger A.; Beck-Sickinger, Annette G.; Huster, Daniel

    2017-01-01

    The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40–50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor. PMID:28387359

  13. Phenotype expression of gingival fibroblasts cultured on membranes used in guided tissue regeneration.

    PubMed

    Locci, P; Calvitti, M; Belcastro, S; Pugliese, M; Guerra, M; Marinucci, L; Staffolani, N; Becchetti, E

    1997-09-01

    Human gingival fibroblasts were cultured in vitro using as substrates an extracellular matrix (matrix) and polytetrafluoride (PTFE) membranes, which are used in guided tissue regeneration. To test the degree of biocompatibility of these membranes, the cellular proliferation and the accumulation of extracellular matrix (ECM) macromolecules were considered as parameters. The fibroblasts were cultured in vitro for 24 and 48 hours without serum on plastic, matrix, and PTFE membranes in the presence of 3H-thymidine, 3H-glucosamine, and 3H-proline to study the neo-synthesis of DNA, glycosaminoglycans (GAG), and collagen proteins, respectively. Studies on cell proliferation showed that fibroblasts grown on matrix membrane significantly increased 3H-thymidine incorporation, while fibroblasts grown on PTFE membrane decreased 3H-thymidine incorporation, compared to plastic used as a control. Moreover, the PTFE membrane induced a marked decrease of collagen and GAG accumulation both in the cellular and extracellular pool, while the matrix membrane provoked a decrease of the two macromolecules in the cellular pool and an increase in the extracellular one, compared to the control. The data we obtained demonstrate that matrix membranes are the most suitable to stimulate both cellular proliferation and ECM macromolecule accumulation.

  14. Functional expression, purification, characterization, and membrane reconstitution of non-structural protein 2 from hepatitis C virus.

    PubMed

    Fogeron, Marie-Laure; Paul, David; Jirasko, Vlastimil; Montserret, Roland; Lacabanne, Denis; Molle, Jennifer; Badillo, Aurélie; Boukadida, Célia; Georgeault, Sonia; Roingeard, Philippe; Martin, Annette; Bartenschlager, Ralf; Penin, François; Böckmann, Anja

    2015-12-01

    Non-structural protein 2 (NS2) of the hepatitis C virus (HCV) is an integral membrane protein that contains a cysteine protease and that plays a central organizing role in assembly of infectious progeny virions. While the crystal structure of the protease domain has been solved, the NS2 full-length form remains biochemically and structurally uncharacterized because recombinant NS2 could not be prepared in sufficient quantities from cell-based systems. We show here that functional NS2 in the context of the NS2-NS3pro precursor protein, ensuring NS2-NS3 cleavage, can be efficiently expressed by using a wheat germ cell-free expression system. In this same system, we subsequently successfully produce and purify milligram amounts of a detergent-solubilized form of full-length NS2 exhibiting the expected secondary structure content. Furthermore, immuno-electron microscopy analyses of reconstituted proteoliposomes demonstrate NS2 association with model membranes.

  15. Analysis of P-glycoprotein expression in purified parasite plasma membrane and food vacuole from Plasmodium falciparum.

    PubMed

    Elandaloussi, Laurence M; Lindt, Meinrad; Collins, Malcolm; Smith, Peter J

    2006-11-01

    A P-glycoprotein homologue (Pgh1) is believed to play a role in modulating levels of chloroquine resistance in Plasmodium falciparum. To study the role of Pgh1 in the mechanism of chloroquine (CQ) resistance, antisera were raised against this protein. There was no direct association between the level of Pgh1 expression and chloroquine sensitivity. We also failed to detect phosphorylation of Pgh1 in the food vacuole (FV), suggesting that other mechanisms regulate the chloroquine-resistant (CQR) phenotype. Therefore, high levels of expression of Pgh1 or phosphorylation of this protein in the FV could not account for CQ sensitivity. In addition, the lack of inhibition of CQ accumulation by anti-Pgh1 antibodies suggests that Pgh1 is not involved as a CQ transporter in the plasma membrane of P. falciparum. Furthermore, resistance reversers do not appear to act at the plasma membrane level.

  16. Focus on membrane differentiation and membrane domains in the prokaryotic cell.

    PubMed

    Boekema, Egbert J; Scheffers, Dirk-Jan; van Bezouwen, Laura S; Bolhuis, Henk; Folea, I Mihaela

    2013-01-01

    A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology

  17. Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complex.

    PubMed

    Nicchia, Grazia Paola; Cogotzi, Laura; Rossi, Andrea; Basco, Davide; Brancaccio, Andrea; Svelto, Maria; Frigeri, Antonio

    2008-06-01

    Altered aquaporin-4 (AQP4) expression has been reported in brain edema, tumors, muscular dystrophy, and neuromyelitis optica. However, the plasma membrane organization of AQP4 and its interaction with proteins such as the dystrophin-associated protein complex are not well understood. In this study, we used sucrose density gradient ultracentrifugation and 2D blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the expression of several AQP4 multi-subunit complexes (pools) of different sizes, ranging from > 1 MDa to approximately 500 kDa and containing different ratios of the 30/32 kDa AQP4 isoforms, indicative of orthogonal arrays of particles of various sizes. A high molecular weight pool co-purified with dystrophin and beta-dystroglycan and was drastically reduced in the skeletal muscle of mdx3cv mice, which have no dystrophin. The number and size of the AQP4 pools were the same in the kidney where dystrophin is not expressed, suggesting the presence of dystrophin-like proteins for their expression. We found that AQP2 is expressed only in one major pool of approximately 500 kDa, indicating that the presence of different pools is a peculiarity of AQP4 rather than a widespread feature in the AQP family. Finally, in skeletal muscle caveolin-3 did not co-purify with any AQP4 pool, indicating the absence of interaction of the two proteins and confirming that caveolae and orthogonal arrays of particles are two independent plasma membrane microdomains. These results contribute to a better understanding of AQP4 membrane organization and raise the possibility that abnormal expression of specific AQP4 pools may be found in pathological states.

  18. Membrane immunoglobulin expressed by retroviral vector gene transfer mimics partial function of the B-cell receptor in vivo.

    PubMed

    Lu, Jing; Chen, Feng; Xu, Zhen; Zhang, Lingling; Xu, Peng; Liu, Depei; Liang, Chihchuan

    2016-01-01

    Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell receptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, resulting in the phosphorylation of Syk and Erk1/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.

  19. 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.

  20. The `heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the gene, nucleotide and amino acid sequence

    PubMed Central

    Michel, H.; Weyer, K. A.; Gruenberg, H.; Lottspeich, F.

    1985-01-01

    The gene coding for the `heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis was isolated in an expression vector. Expression of the heavy subunit in Escherichia coli was detected with antibodies raised against crystalline reaction centres. The entire subunit, and not a fusion protein, was expressed in E. coli. The protein coding region of the gene was sequenced and the amino acid sequence derived. Part of the amino acid sequence was confirmed by chemical sequence analysis of the protein. The heavy subunit consists of 258 amino acids and its mol. wt. is 28 345. It possesses one membrane-spanning α-helical segment, as was revealed by the concomitant X-ray structure analysis. ImagesFig. 1.Fig. 2. PMID:16453623

  1. Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

    PubMed Central

    Jones, L V; Compans, R W; Davis, A R; Bos, T J; Nayak, D P

    1985-01-01

    We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process. Images PMID:3016520

  2. Expression of membrane-type 1 matrix metalloproteinase and activation of progelatinase A in human osteoarthritic cartilage.

    PubMed Central

    Imai, K.; Ohta, S.; Matsumoto, T.; Fujimoto, N.; Sato, H.; Seiki, M.; Okada, Y.

    1997-01-01

    Matrix metalloproteinases (MMPs) are expressed in osteoarthritic (OA) cartilage and are thought to be involved in the degradation of cartilage extracellular matrix (ECM). Among these proteinases, MMP-2 (gelatinase A) demonstrates a wide range of substrate specificity against the ECM present in cartilage. Although MMP-2 expression increases in OA cartilage, the activation mechanism of the corresponding zymogen (pro-MMP-2) in cartilage is unknown. In this study, we examined the expression pattern of membrane-type 1 MMP (MT1-MMP) in human OA articular cartilage and its correlation with the activation of pro-MMP-2. Immunohistochemical studies demonstrate that MT1-MMP localizes to the chondrocytes in the superficial and transitional zones in all of the samples examined directly correlating with cartilage degradation. Reverse transcription polymerase chain reaction confirmed the predominant expression of MT1-MMP mRNA in the OA cartilage. In situ hybridization revealed the site of expression of MT1-MMP in OA cartilage to be the chondrocytes. Through gelatin zymography and a sandwich enzyme immunoassay it was demonstrated that OA cartilage explants secrete significantly higher levels of pro-MMP-2 than normal samples. Pro-MMP-2 activation was enhanced in the OA cartilage samples and correlated with MT1-MMP expression in the cartilage. Plasma membranes prepared from cultured chondrocytes with MT1-MMP expression and those directly isolated from OA cartilage could activate pro-MMP-2. MT1-MMP gene expression in cultured chondrocytes was induced by treatment with interleukin-1 alpha and/or tumor necrosis factor-alpha. These data suggest that cytokine-induced MT1-MMP in the chondrocytes may play a key role in the activation of pro-MMP-2 in the OA articular cartilage, leading to cartilage destruction through ECM degradation. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 PMID:9212749

  3. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    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 LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were 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 eight 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(LN-229) column and decreased it (-5%) on the NMAC(LN-229), 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.

  4. Differential expression profile of membrane proteins in Aplysia pleural–pedal ganglia under the stress of methyl parathion.

    PubMed

    Chen, Ying-Ying; Huang, Lin; Zhang, Yong; Ke, Cai-Huan; Huang, He-Qing

    2014-03-01

    This study was aimed to analyze the alteration of membrane protein profiles in Aplysia juliana Quoy & Gaimard (A. juliana) pleural–pedal ganglia under MP exposure. Both the results of GC–MS analysis and the activity assay of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT) reveal that MP toxicological effects on Aplysia left and right pleural–pedal ganglia are different under 7 and 14 days of exposure. Therefore, Aplysia were subjected for exposure at two concentrations (1 and 2 mg/l) of MP for 7 and 14 days for membrane proteomic study. As a result, 19 and 14 protein spots were differentially expressed in A. juliana left pleural–pedal ganglia under 7 and 14 days treatment, and 20 and 14 protein spots found with differential expressions in their right ganglia under the same treatment, respectively. Several proteins with expression variations were detected from both the left and right pleural–pedal ganglia; however, most proteins have distinctive expressions, indicating different mechanisms might be involved in initiating MP toxicology in left and right ganglia. Among the total differential protein spots obtained, 29 proteins were classed as membrane proteins. These proteins are mainly involved in the metabolism process, cell redox homeostasis, signal transduction, immunology, intracellular transport and catalysis, indicating MP toxicity in mollusks seems to be complex and diverse. Some differentially expressed proteins were further confirmed by Western blotting and quantitative real-time PCR. These results might provide renovated insights to reveal the mechanism of MP-induced neurotoxicity, and the novel candidate biomarkers might have potential application for environmental evaluation of MP pollution level.

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

    PubMed

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

    2013-11-01

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

  6. 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

  7. Plasmidless, photosynthetically incompetent mutants of Rhodospirillum rubrum.

    PubMed Central

    Kuhl, S A; Wimer, L T; Yoch, D C

    1984-01-01

    Ethyl methanesulfonate rendered a high percentage of Rhodospirillum rubrum cells plasmidless and photosynthetically incompetent (Kuhl et al., J. Bacteriol. 156:737-742, 1983). By probing restriction endonuclease-digested chromosomal DNA from these plasmidless strains with 32P-labeled R. rubrum plasmid DNA, we showed that no homology exists between the plasmid and the chromosomal DNA of the mutant. Loss of the plasmid in all the nonphotosynthetic isolates was accompanied by the synthesis of spirilloxanthin under aerobic growth conditions, resistance to cycloserine and HgCl2, and loss of ability to grow fermentatively on fructose. Changes in both the protein and lipid composition of the membranes and the impaired uptake of 203HgCl2 in the plasmidless strains (compared with the wild type) suggest either that membrane modification occurs as a result of plasmid loss, accounting for several of the acquired phenotype characteristics of the cured strains, or that both membrane modification and plasmid loss are part of the same pleiotropic mutation. Images PMID:6434514

  8. 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

  9. Differential allocation to photosynthetic and non-photosynthetic nitrogen fractions among native and invasive species.

    PubMed

    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.

  10. Increased expression of plasma membrane Ca(2+)ATPase 4b in platelets from hypertensives: a new sign of abnormal thrombopoiesis?

    PubMed

    Dally, Saoussen; Chaabane, Chiraz; Corvazier, Elisabeth; Bredoux, Raymonde; Bobe, Regis; Ftouhi, Bochra; Slimane, Hedia; Raies, Aly; Enouf, Jocelyne

    2007-11-01

    Platelet Ca(2+) homeostasis is controlled by a multi-Ca(2+)ATPase system including two PMCA (plasma membrane Ca(2+)ATPase) and seven SERCA (sarco/endoplasmic reticulum Ca(2+)ATPase) isoforms. Previous studies have shown similar platelet Ca(2+) abnormalities in diabetic and hypertensive patients, including an increase in intracellular [Ca(2+)](I), a possible modulation of PMCA activity and increased PMCA tyrosine phosphorylation. Very recently, we found that platelets from diabetic patients also exhibited increased PMCA4b expression. In the present study we looked for further similarities between diabetic and hypertensive patients. We first confirmed a decrease in Ca(2+)ATPase activity (mean 55 + 7%) in mixed platelet membranes isolated from 10 patients with hypertension compared with those from 10 healthy controls. In addition, the decreased Ca(2+)ATPase activity correlated with the DBP of the different patients, as expected for PMCA activity. Second, we performed a pilot study of six hypertensives to examine their expressions of PMCA and SERCA mRNA and proteins. Like the diabetic patients, 100% of hypertensives were found to present a major increase in PMCA4b expression (mean value of 218 +/- 21%). We thus determined that platelets from diabetic and hypertensive patients showed similar increased PMCA4b isoform. Since increased PMCA4b expression was recently found to be associated with a perturbation of megakaryocytopoiesis, these findings may also point to an abnormality in platelet maturation in hypertension.

  11. 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

  12. Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL).

    PubMed Central

    Tabilio, A; Rosa, J P; Testa, U; Kieffer, N; Nurden, A T; Del Canizo, M C; Breton-Gorius, J; Vainchenker, W

    1984-01-01

    We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6201359

  13. The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane.

    PubMed

    Re, Michelle; Pampillo, Macarena; Savard, Martin; Dubuc, Céléna; McArdle, Craig A; Millar, Robert P; Conn, P Michael; Gobeil, Fernand; Bhattacharya, Moshmi; Babwah, Andy V

    2010-07-08

    The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.

  14. ADAM12 is expressed in the tumour vasculature and mediates ectodomain shedding of several membrane-anchored endothelial proteins.

    PubMed

    Fröhlich, Camilla; Klitgaard, Marie; Noer, Julie B; Kotzsch, Alexander; Nehammer, Camilla; Kronqvist, Pauliina; Berthelsen, Jens; Blobel, Carl; Kveiborg, Marie; Albrechtsen, Reidar; Wewer, Ulla M

    2013-05-15

    ADAM (a disintegrin and metalloproteinase) 12 is a metalloprotease implicated in cancer progression. ADAM12 can activate membrane-anchored proteins, such as sonic hedgehog, Delta-like 1 and certain epidermal growth factor receptor ligands, through a process called ectodomain shedding. We screened several membrane-anchored proteins to further dissect the substrate profile of ADAM12-mediated ectodomain shedding, and found shedding of five previously unreported substrates [Kitl1, VE-cadherin (vascular endothelial cadherin), Flk-1 (fetal liver kinase 1), Tie-2, and VCAM-1 (vascular cell adhesion molecule 1)], of which the latter four are specifically expressed by endothelial cells. We also observed that ADAM12 expression was increased in the tumour vasculature of infiltrating ductal carcinoma of the human breast as compared with little to no expression in normal breast tissue vasculature, suggesting a role for ADAM12 in tumour vessels. These results prompted us to further evaluate ADAM12-mediated shedding of two endothelial cell proteins, VE-cadherin and Tie-2. Endogenous ADAM12 expression was very low in cultured endothelial cells, but was significantly increased by cytokine stimulation. In parallel, the shed form of VE-cadherin was elevated in such cytokine-stimulated endothelial cells, and ADAM12 siRNA (small interfering RNA) knockdown reduced cytokine-induced shedding of VE-cadherin. In conclusion, the results of the present study demonstrate a role for ADAM12 in ectodomain shedding of several membrane-anchored endothelial proteins. We speculate that this process may have importance in tumour neovascularization or/and tumour cell extravasation.

  15. Diabetes increases facilitative glucose uptake and GLUT2 expression at the rat proximal tubule brush border membrane

    PubMed Central

    Marks, Joanne; Carvou, Nicolas J C; Debnam, Edward S; Srai, Surjit K; Unwin, Robert J

    2003-01-01

    The mechanism of renal glucose transport involves the reabsorption of filtered glucose from the proximal tubule lumen across the brush border membrane (BBM) via a sodium-dependent transporter, SGLT, and exit across the basolateral membrane via facilitative, GLUT-mediated, transport. The aim of the present study was to determine the effect of streptozotocin-induced diabetes on BBM glucose transport. We found that diabetes increased facilitative glucose transport at the BBM by 67.5 % (P < 0.05) – an effect that was abolished by overnight fasting. Western blotting and immunohistochemistry demonstrated GLUT2 expression at the BBM during diabetes, but the protein was undetectable at the BBM of control animals or diabetic animals that had been fasted overnight. Our findings indicate that streptozotocin-induced diabetes causes the insertion of GLUT2 into the BBM and this may provide a low affinity/high capacity route of entry into proximal tubule cells during hyperglycaemia. PMID:12963802

  16. Diabetes increases facilitative glucose uptake and GLUT2 expression at the rat proximal tubule brush border membrane.

    PubMed

    Marks, Joanne; Carvou, Nicolas J C; Debnam, Edward S; Srai, Surjit K; Unwin, Robert J

    2003-11-15

    The mechanism of renal glucose transport involves the reabsorption of filtered glucose from the proximal tubule lumen across the brush border membrane (BBM) via a sodium-dependent transporter, SGLT, and exit across the basolateral membrane via facilitative, GLUT-mediated, transport. The aim of the present study was to determine the effect of streptozotocin-induced diabetes on BBM glucose transport. We found that diabetes increased facilitative glucose transport at the BBM by 67.5 % (P < 0.05)--an effect that was abolished by overnight fasting. Western blotting and immunohistochemistry demonstrated GLUT2 expression at the BBM during diabetes, but the protein was undetectable at the BBM of control animals or diabetic animals that had been fasted overnight. Our findings indicate that streptozotocin-induced diabetes causes the insertion of GLUT2 into the BBM and this may provide a low affinity/high capacity route of entry into proximal tubule cells during hyperglycaemia.

  17. Conserved outer membrane protein of Neisseria meningitidis involved in capsule expression.

    PubMed Central

    Frosch, M; Müller, D; Bousset, K; Müller, A

    1992-01-01

    In Neisseria meningitidis, translocation of capsular polysaccharides to the cell surface is mediated by a transport system that fits the characteristics of ABC (ATP-binding cassette) transporters. One protein of this transport system, termed CtrA, is located in the outer membrane. By use of a CtrA-specific monoclonal antibody, we could demonstrate that CtrA occurs exclusively in N. meningitidis and not in other pathogenic or nonpathogenic Neisseria species. Nucleotide sequence comparison of the ctrA gene from different meningococcal serogroups indicated that CtrA is strongly conserved in all meningococcal serogroups, independent of the chemical composition of the capsular polysaccharide. Secondary structure analysis revealed that CtrA is anchored in the outer membrane by eight membrane-spanning amphipathic beta strands, a structure of proteins that function as porins. Images PMID:1371768

  18. Approximate expression for the potential energy of the double-layer interaction between two parallel ion-penetrable membranes at small separations in an electrolyte solution.

    PubMed

    Ohshima, Hiroyuki

    2010-10-01

    An approximate expression for the potential energy of the double-layer interaction between two parallel similar ion-penetrable membranes in a symmetrical electrolyte solution is derived via a linearization method, in which the nonlinear Poisson-Boltzmann equations in the regions inside and outside the membranes are linearized with respect to the deviation of the electric potential from the Donnan potential. This approximation works quite well for small membrane separations h for all values of the density of fixed charges in the membranes (or the Donnan potential) and gives a correct limiting form of the interaction energy (or the interaction force) as h-->0.

  19. Use of polymeric membranes for purification of an E. coli expressed biotherapeutic protein.

    PubMed

    Muthukumar, S; Rathore, Anurag S

    2016-01-01

    Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed-bed chromatography by eliminating the diffusion-related limitations associated with the traditional resin beads. In this article, we examine six membrane adsorbers for purification of granulocyte colony-stimulating factor (GCSF), an Escherichia coli-based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the article.

  20. Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human fetal membranes throughout pregnancy and at term.

    PubMed

    Brown, N L; Slater, D M; Alvi, S A; Elder, M G; Sullivan, M H; Bennett, P R

    1999-07-01

    Lipoxygenase metabolites may be involved in human parturition. 5-lipoxygenase (5-LOX) catalyses the first steps in the synthesis of leukotrienes from arachidonic acid, and its activity is dependent on 5-LOX activating protein (FLAP). The expression of 5-LOX and FLAP were investigated in fetal membranes to determine whether there are changes with gestational age or at term with the onset of labour. No significant differences were found in the expression of 5-LOX or FLAP mRNA in the amnion at different gestational ages or at term. In the chorion-decidua, 5-LOX mRNA expression was significantly higher in the first trimester of pregnancy than in the second and third trimesters. At term, there was a significant increase in both 5-LOX mRNA and protein expression in the chorion-decidua in the time after labour, compared with the time before labour. The expression of FLAP mRNA was also significantly higher in the chorion-decidua in the first trimester of pregnancy compared with the third trimester, and at term in the time after labour compared with the time before labour. Expression of FLAP protein was not studied, as an antibody is not currently available. These results are consistent with a role for 5-LOX and FLAP in the control of parturition at term, and also suggest an involvement earlier in pregnancy.

  1. Progesterone receptor membrane component 1 (PGRMC1) gene expression in corpus luteum during the estrous cycle in cows.

    PubMed

    Kowalik, Magdalena K; Kotwica, Jan

    2008-11-01

    The aim of the study was to investigate progesterone receptor membrane component 1 (PGRMC1) mRNA expression in bovine corpus luteum (CL) obtained from heifers or non-pregnant cows on the following days of the estrous cycle: 1-5, 6-10, 11-16 and 17-21 (n=4/each time period). The expression of PGRMC1 mRNA, analyzed by semiquantitative RT-PCR, was the highest on days 6-10 (p<0.01) and then it declined (p<0.05). The lowest expression was found on days 1-5 (p<0.05). A significant correlation (p<0.05) was also observed between luteal progesterone (P(4)) concentration and PGRMC1 mRNA expression. These data indicate that PGRMC1 mRNA is expressed in bovine CL and this expression varies throughout the luteal phase. It is assumed that PGRMC1 may be involved in a non-genomic effect of P(4) on luteal cells.

  2. Photoperiodic regulation of melatonin membrane receptor (MT1R) expression and steroidogenesis in testis of adult golden hamster, Mesocricetus auratus.

    PubMed

    Mukherjee, Arun; Haldar, Chandana

    2014-11-01

    Photoperiodic modulation of melatonin membrane receptor (MT1R) expression in testis has never been reported for any seasonal breeder. Thus, the aim of the present study was to investigate the expression dynamics of MT1R in testis and its interaction with testicular steroidogenesis in a long-day breeder, Mesocricetus auratus. Hamsters were exposed to different photoperiodic conditions i.e. critical- (CP; 12.5L:11.5D); short-day- (SD; 8L:16D) and long-day- (LD; 16L:8D) for 10 weeks wherein testicular steroidogenesis, local melatonin synthesis and the expression of MT1R were analyzed. SD induced melatonin suppressed testicular steroidogenesis as evident from regressed testicular histoarchitecture, decreased expression of AR, StAR, LH-R, P₄₅₀SCC and enzyme activities of 3β- and 17β-HSD. Differential photoperiodic regulation of MT1R expression in testis suggests its involvement in photoperiodic signal transduction for seasonal adjustment of reproduction. Increased S-NAT (Serotonin N-acetyl transferase) activity and local testicular melatonin under SD condition suggest an inhibitory effect of the local melatonergic system on testicular steroidogenesis. Completely opposite responses were recorded for all the parameters analyzed when hamsters were exposed to CP or LD conditions. In conclusion, we may suggest that photoperiod via regulating circulatory and local melatonin level as well as MT1R expression in testes fine tunes the steroidogenesis and thereby, the reproductive status of male golden hamster.

  3. Optimization of Light-Harvesting Pigment Improves Photosynthetic Efficiency.

    PubMed

    Jin, Honglei; Li, Mengshu; Duan, Sujuan; Fu, Mei; Dong, Xiaoxiao; Liu, Bing; Feng, Dongru; Wang, Jinfa; Wang, Hong-Bin

    2016-11-01

    Maximizing light capture by light-harvesting pigment optimization represents an attractive but challenging strategy to improve photosynthetic efficiency. Here, we report that loss of a previously uncharacterized gene, HIGH PHOTOSYNTHETIC EFFICIENCY1 (HPE1), optimizes light-harvesting pigments, leading to improved photosynthetic efficiency and biomass production. Arabidopsis (Arabidopsis thaliana) hpe1 mutants show faster electron transport and increased contents of carbohydrates. HPE1 encodes a chloroplast protein containing an RNA recognition motif that directly associates with and regulates the splicing of target RNAs of plastid genes. HPE1 also interacts with other plastid RNA-splicing factors, including CAF1 and OTP51, which share common targets with HPE1. Deficiency of HPE1 alters the expression of nucleus-encoded chlorophyll-related genes, probably through plastid-to-nucleus signaling, causing decreased total content of chlorophyll (a+b) in a limited range but increased chlorophyll a/b ratio. Interestingly, this adjustment of light-harvesting pigment reduces antenna size, improves light capture, decreases energy loss, mitigates photodamage, and enhances photosynthetic quantum yield during photosynthesis. Our findings suggest a novel strategy to optimize light-harvesting pigments that improves photosynthetic efficiency and biomass production in higher plants.

  4. Human melanoma cells express FGFR/Src/Rho signaling that entails an adhesion-independent caveolin-1 membrane association.

    PubMed

    Fecchi, Katia; Travaglione, Sara; Spadaro, Francesca; Quattrini, Adriano; Parolini, Isabella; Piccaro, Giovanni; Raggi, Carla; Fabbri, Alessia; Felicetti, Federica; Carè, Alessandra; Fiorentini, Carla; Sargiacomo, Massimo

    2012-03-15

    Caveolae have been indicated as a center of cytoskeleton regulation for Src kinase/Rho GTPase signaling. In addition, Src recruitment on intact cortical actin cytoskeleton appears to be required for bFGF/FGFR signal activation. Recently, we established a relationship between caveolin-1 (Cav-1) expression and cell migration in human malignant melanoma, constitutively activated by a bFGF autoregulatory loop. This work intends to investigate whether caveolae's asset, through bFGF/FGFR/c-Src/Rho signaling, could be related to melanoma cell anchorage. Accordingly, we revealed the existence of a FGFR/Src kinase pathway in Cav-1 enriched detergent-resistant membranes (DRMs) of Me665/1 metastatic melanoma cells, as confirmed by FGFR silencing. Moreover, we determined the expression and phosphorylation levels of Cav-1/Src/Erk signal pathway as a function of FGFR activation and cell density. A sucrose density gradient ultracentrifugation was employed to monitor Cav-1 membrane association and buoyancy in Me665/1 cells treated for actin fragmentation or for altered phosphorylation signals. As a result, melanoma cells show remarkable resistance to Cav-1 disassembly, together with persisting cell signal activity, being Src and Cav-1 crucial modulators of Rho GTPases. In conclusion, our study primarily highlights, in a metastatic melanoma cell line expressing caveolin, the circumstances whereby caveola structural and functional endurance enables the FGFR/Src/Rho GTPases pathway to keep on cell progression.

  5. Function and Expression of an N-Acetylneuraminic Acid-Inducible Outer Membrane Channel in Escherichia coli

    PubMed Central

    Condemine, Guy; Berrier, Catherine; Plumbridge, Jacqueline; Ghazi, Alexandre

    2005-01-01

    The Escherichia coli yjhA (renamed nanC) gene encodes a protein of the KdgM family of outer membrane-specific channels. It is transcribed divergently from fimB, a gene involved in the site-specific inversion of the region controlling transcription of the fimbrial structural genes but is separated from it by one of the largest intergenic regions in E. coli. We show that nanC expression is induced by N-acetylneuraminic acid and modulated by N-acetylglucosamine. This regulation occurs via the NanR and NagC regulators, which also control fimB expression. nanC expression is also activated by the regulators cyclic AMP-catabolite activator protein, OmpR, and CpxR. When the NanC protein was reconstituted into liposomes, it formed channels with a conductance of 450 pS at positive potential and 300 to 400 pS at negative potential in 800 mM KCl. The channels had a weak anionic selectivity. In an ompR background, where the general porins OmpF and OmpC are absent, NanC is required for growth of E. coli on N-acetylneuraminic acid as the sole carbon source. All these results suggest that NanC is an N-acetylneuraminic acid outer membrane channel protein. PMID:15743943

  6. Transcriptional analysis of in vitro expression patterns of Chlamydophila abortus polymorphic outer membrane proteins during the chlamydial developmental cycle

    PubMed Central

    Wheelhouse, Nicholas; Aitchison, Kevin; Spalding, Lucy; Livingstone, Morag; Longbottom, David

    2009-01-01

    Chlamydophila abortus is the aetiological agent of ovine enzootic abortion. Sequencing, annotation and comparative analysis of the genome of C. abortus strain S26/3 has revealed variation in the loci encoding the polymorphic membrane proteins (Pmps). These Pmps resemble autotransporter proteins of the type V secretion system, suggesting an important role in chlamydial pathogenesis. The purpose of this study was to characterise the transcriptional expression patterns of this family during the developmental cycle of C. abortus. McCoy cells were infected with C. abortus and analysed for pmp mRNA expression over a 72 h period. Few pmp transcripts were detected in the early stages of the developmental cycle. Peak expression occurred at 48 h post-infection (p.i.) other than for pmp5E, where it was observed at 24 h p.i. Overall, expression of pmps 5E, 18D and 10G were found to be 40 to 100-fold higher than the lowest expressing pmps (6H, 13G and 15G) at 24 h p.i., while pmps 18D and 17G were 14 to 16-fold higher than the lowest (11G, 14G and 15G) at 48 h. Levels of expression for all the other pmp genes were below one copy per genome at any time point. The expression of all the pmps reduced to near base-line levels by 60 h p.i. These results demonstrate that pmp expression in C. abortus is mid to late cycle, consistent with conversion of the reticulate body to the elementary body. The low level of pmp transcription may be indicative of heterogeneity in expression, suggesting a possible role for some of the Pmps in antigenic variation and chlamydial pathogenesis. PMID:19454212

  7. 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.

  8. 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.

  9. 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.

  10. Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

    PubMed Central

    Thelin, William R.; Chen, Yun; Gentzsch, Martina; Kreda, Silvia M.; Sallee, Jennifer L.; Scarlett, Cameron O.; Borchers, Christoph H.; Jacobson, Ken; Stutts, M. Jackson; Milgram, Sharon L.

    2007-01-01

    The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation. PMID:17235394

  11. Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs.

    PubMed

    Sancho, S; Casas, I; Ekwall, H; Saravia, F; Rodriguez-Martinez, H; Rodriguez-Gil, J E; Flores, E; Pinart, E; Briz, M; Garcia-Gil, N; Bassols, J; Pruneda, A; Bussalleu, E; Yeste, M; Bonet, S

    2007-07-01

    This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the 'Entrepelado' and 'Lampiño' breeds. The samples were suspended in a commercial extender and refrigerated to 17 degrees C for transport to the laboratory (step A), where they were further extended with a lactose-egg yolk-based extender and chilled to 5 degrees C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS-PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the 'Entrepelado' breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing-thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.

  12. Eimeria bovis meront I-carrying host cells express parasite-specific antigens on their surface membrane.

    PubMed

    Badawy, Ahmed Ibrahem I; Lutz, Kathleen; Taubert, Anja; Zahner, Horst; Hermosilla, Carlos

    2010-02-01

    Host immune responses conducted against antigens of Eimeria bovis are key factors for the development of protective immunity against this protozoan disease. In this study we investigated the expression of E. bovis-derived antigens on the host cell surface membrane during E. bovis first merogony in vitro. Host cells carrying E. bovis-meront I stages expressed E. bovis host cell surface antigens (EbHCSAg) on their surface membrane which were recognised by hyperimmune sera of calves and by sera from rats immunized with E. bovis merozoites I, when tested by indirect immune fluorescent antibody test (IIFAT), laser scanning confocal microscopy (LSCM) and immune electron microscopy. Expression of EbHCSAg on permissive host cells was earliest detected 7 days p. i., thus coinciding with the onset of the parasite replication. Membrane-associated EbHCSAg were removed from infected host cells by proteinase K, partially by Triton X-100, Triton X-114 and Triton X-405, but not by 1 M NaCl, CHAPS or phospholipase C treatment. Antibodies, affinity-purified on paraformaldehyde/glutardialdehyde (PAGA)-fixed E. bovis meront I-infected bovine host cells bound to the surface meront I-carrying cells and to merozoites I (IIFAT, LSCM) but, in contrast to untreated sera, not to sporozoites. When tested on methanol-fixed merozoites I and sporozoites by IIFAT, affinity-purified antibodies bound to structures in the apical complex area of merozoites I, but not to sporozoites, whilst untreated sera caused diffuse labelling of internal structures of both parasite stages. Immune electron microscopy demonstrated binding of affinity-purified antibodies to micronemes and dense granules of merozoites I. Although the function of EbHCSAg is still unknown, results of this study might suggest an involvement in the development of protective immunity against E. bovis infections.

  13. Human bronchial smooth muscle cells express adenylyl cyclase isoforms 2, 4, and 6 in distinct membrane microdomains.

    PubMed

    Bogard, Amy S; Xu, Congfeng; Ostrom, Rennolds S

    2011-04-01

    Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because β-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). Reverse transcriptase-polymerase chain reaction and immunoblot analyses detected expression of AC2, AC4, and AC6. Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca(2+)-inhibited and G(βγ)-stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca(2+) but unaltered by G(βγ), whereas butaprost-stimulated AC activity was stimulated by G(βγ) but unaffected by Ca(2+) addition. Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions, whereas AC2 and AC4 localized in nonraft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca(2+)-inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G(βγ)-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E(2) or butaprost. β(2)AR, but not prostanoid EP(2) or EP(4) receptors, colocalized with AC5/6 in lipid raft fractions. Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization in membrane microdomains. These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca(2+) and G(βγ).

  14. Environmental control of photosynthetic enhancement.

    PubMed

    Punnett, T

    1971-01-22

    The transition from granular to homogeneous chloroplasts in vivo in Egeria densa caused by environmental conditions was paralleled by a decrease in photosynthetic enhancement from 30 percent to nearly zero. The drop in enhancement can be explained either by a change in the partitioning of light energy between the two photosystems or a change to a single photosystem.

  15. 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...

  16. A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase.

    PubMed

    Pedro, A Q; Bonifácio, M J; Queiroz, J A; Maia, C J; Passarinha, L A

    2011-11-10

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37°C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.

  17. Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32

    PubMed Central

    Eshghi, Azad; Pinne, Marija; Haake, David A.; Zuerner, Richard L.; Frank, Ami

    2012-01-01

    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 immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence. PMID:22174381

  18. Comparative analysis of fibrillar and basement membrane collagen expression in embryos of the sea urchin, Strongylocentrotus purpuratus.

    PubMed

    Suzuki, H R; Reiter, R S; D'Alessio, M; Di Liberto, M; Ramirez, F; Exposito, J Y; Gambino, R; Solursh, M

    1997-06-01

    The time of appearance and location of three distinct collagen gene transcripts termed 1 alpha, 2 alpha, and 3 alpha, were monitored in the developing S. purpuratus embryo by in situ hybridization. The 1 alpha and 2 alpha transcripts of fibrillar collagens were detected simultaneously in the primary (PMC) and secondary (SMC) mesenchyme cells of the late gastrula stage and subsequently expressed in the spicules and gut associated cells of the pluteus stage. The 3 alpha transcripts of the basement membrane collagen appeared earlier than 1 alpha and 2 alpha, and were first detected in the presumptive PMC at the vegetal plate of the late blastula stage. The PMC exhibited high expression of 3 alpha at the mesenchyme blastula stage, but during gastrulation the level of expression was reduced differentially among the PMC. In the late gastrula and pluteus stages, both PMC and SMC expressed 3 alpha mRNA, and thus at these stages all three collagen genes displayed an identical expression pattern by coincidence. This study thus provides the first survey of onset and localization of multiple collagen transcripts in a single sea urchin species.

  19. Differential expression profile of membrane proteins in zebrafish (Danio rerio) brain exposed to methyl parathion.

    PubMed

    Huang, Qing-Yu; Huang, He-Qing

    2011-09-01

    Methyl parathion (MP) is a widely used organophosphorus pesticide, which has been related to a broad spectrum of toxic effects on environmental organisms. The present study investigated the changes in the protein profile of enriched membrane fraction from zebrafish (Danio rerio) brain exposed to three concentrations (0.5, 1 and 2 mg/L) of MP. 2-DE revealed that the abundance of 21 protein spots was significantly changed by MP stress. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database search, 16 protein spots were identified as membrane proteins, among which 8 were down-regulated, while 8 were up-regulated. These proteins are mainly involved in oxidative stress response, signal transduction, metabolism, protein synthesis and degradation, neuroplasticity and regeneration as well as synaptic transmission. These results may aid our understanding of the mechanism of MP-induced neurotoxicity and provide the possibility of the establishment of candidate biomarkers of MP.

  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. Acute intrastriatal administration of quinolinic acid affects the expression of the coat protein AP-2 and its interaction with membranes.

    PubMed

    Borgonovo, Janina; Seltzer, Alicia; Sosa, Miguel Angel

    2009-10-01

    Clathrin-coated vesicle endocytosis is thought to be crucial for the maintenance of synaptic transmission and for the cell plasticity at the nervous system. In this study, we demonstrated that acute intrastriatal administration of quinolinic acid (QUIN), an agonist of the N-methyl-D: -aspartate receptor, induces a decrease of the coat protein AP-2 expression and affects their interaction with membranes. By western blot analysis we observed that at 24 h after QUIN intrastriatal injection, alpha1 subunit of AP-2 and alpha2, at lesser extent, were reduced in the striatal membranes. The decrease of both subunits expression was extended to 48 h after treatment, although the soluble proteins were mostly affected. Other areas of the brain were not affected by the treatment, except the cerebellum, where a significant increase of soluble AP-2 (both subunits) was observed at 48 h after injection. Another coat protein, as the phosphoprotein AP-180, was not affected by the injection of QUIN. We also confirmed that QUIN injection causes increasing loss of striatal neurons after the administration of the toxin. We concluded that QUIN may affect the endocytotic machinery of the striatum, by inducing changes in the AP-2 behaviour. Consequently, the internalization of NMDAR and/or AMPAR may be affected, by QUIN, contributing to the excitotoxic effect of the drug.

  2. C9ORF135 encodes a membrane protein whose expression is related to pluripotency in human embryonic stem cells

    PubMed Central

    Zhou, Shixin; Liu, Yinan; Ma, Yumin; Zhang, Xiaoyan; Li, Yang; Wen, Jinhua

    2017-01-01

    Human embryonic stem cells (hESCs) are a unique population of cells defined by their capacity for self-renewal and pluripotency. Here, we identified a previously uncharacterized gene in hESCs, C9ORF135, which is sharply downregulated during gastrulation and gametogenesis, along with the pluripotency factors OCT4, SOX2, and NANOG. Human ESCs express two C9ORF135 isoforms, the longer of which encodes a membrane-associated protein, as determined by immunostaining and western blotting of fractionated cell lysates. Moreover, the results of chromatin immunoprecipitation (ChIP), mass spectrometry (MS), and co-immunoprecipitation (co-IP) analyses demonstrated that C9ORF135 expression is regulated by OCT4 and SOX2 and that C9ORF135 interacts with non-muscle myosin IIA and myosin IIB. Collectively, these data indicated that C9ORF135 encodes a membrane-associated protein that may serve as a surface marker for undifferentiated hESCs. PMID:28345668

  3. 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

  4. C9ORF135 encodes a membrane protein whose expression is related to pluripotency in human embryonic stem cells.

    PubMed

    Zhou, Shixin; Liu, Yinan; Ma, Yumin; Zhang, Xiaoyan; Li, Yang; Wen, Jinhua

    2017-03-27

    Human embryonic stem cells (hESCs) are a unique population of cells defined by their capacity for self-renewal and pluripotency. Here, we identified a previously uncharacterized gene in hESCs, C9ORF135, which is sharply downregulated during gastrulation and gametogenesis, along with the pluripotency factors OCT4, SOX2, and NANOG. Human ESCs express two C9ORF135 isoforms, the longer of which encodes a membrane-associated protein, as determined by immunostaining and western blotting of fractionated cell lysates. Moreover, the results of chromatin immunoprecipitation (ChIP), mass spectrometry (MS), and co-immunoprecipitation (co-IP) analyses demonstrated that C9ORF135 expression is regulated by OCT4 and SOX2 and that C9ORF135 interacts with non-muscle myosin IIA and myosin IIB. Collectively, these data indicated that C9ORF135 encodes a membrane-associated protein that may serve as a surface marker for undifferentiated hESCs.

  5. 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.

  6. 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

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

    PubMed

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

    2014-04-01

    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.

  8. Effect of sterol carrier protein-2 expression on sphingolipid distribution in plasma membrane lipid rafts/caveolae.

    PubMed

    Atshaves, Barbara P; Jefferson, John R; McIntosh, Avery L; Gallegos, Adalberto; McCann, Bonnie M; Landrock, Kerstin K; Kier, Ann B; Schroeder, Friedhelm

    2007-10-01

    Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.

  9. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions1

    PubMed Central

    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

    2015-01-01

    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

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

    PubMed

    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

    2014-11-01

    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.

  11. 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

  12. Expression of membrane receptor for tumour necrosis factor on human blood lymphocytes.

    PubMed

    Zola, H; Flego, L; Weedon, H

    1993-08-01

    Using a monoclonal antibody against the human p75 tumour necrosis factor receptor (TNFR-I) combined with a high-sensitivity immunofluorescence flow cytometric procedure, a proportion of peripheral blood lymphocytes can be shown to express TNFR-I constitutively. Approximately 50% of peripheral blood lymphocytes consisting mostly of CD4 cells and including most CD45R0-positive cells, express TNFR-I. Receptor expression is increased by a variety of activation signals. Only a minority (up to 30%) of tonsil B cells express measurable levels of TNFR-I. The tonsil B cells which express TNFR-I include both cells with a germinal centre cell phenotype and cells with the phenotype of the follicular mantle zone. Activation of B cells with anti-immunoglobulin, alone or in combination with interleukin-4 or interleukin-2, increases receptor expression, particularly in cells with the phenotype of mantle zone cells. The functional significance of constitutive expression of TNFR by blood and tissue lymphocytes is discussed.

  13. Expression patterns of progesterone receptor membrane components 1 and 2 in endometria from women with and without endometriosis.

    PubMed

    Bunch, Kristen; Tinnemore, Deborah; Huff, Seth; Hoffer, Zachary S; Burney, Richard O; Stallings, Jonathan D

    2014-02-01

    Endometriosis is a hormone-dependent inflammatory condition associated with pain and infertility. A growing body of evidence supports attenuated secretory-phase progesterone responsiveness in women with this disease. Herein, we compare the expression of progesterone receptor membrane components (PGRMC) 1 and 2 in eutopic endometrium from 11 women with laparoscopically and/or histologically proven stage III/IV endometriosis and 23 disease-free women. Menstrual cycle phase was determined using a combination of reported cycle day, serum hormone profile, and endometrial histologic dating. The PGRMC-1 (fold change -3.3; P < .05) and PGRMC-2 (fold-change -8.8; P < .05) gene expression were significantly downregulated in secretory phase, eutopic endometrium from women with endometriosis. Immunohistochemistry demonstrated decreased PGRMC-1 and PGRMC-2 protein expression in the secretory phase endometrial stroma cells of women with endometriosis. Consistent with the preclinical work of others, our results reflect downregulation of endometrial PGRMC-1 and PGRMC-2 expression in secretory phase endometrium from women with advanced stage endometriosis. Understanding the molecular mechanisms of attenuated progesterone action in endometriosis has important diagnostic and therapeutic implications.

  14. 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.

  15. 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

  16. 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.

  17. Quantum oscillatory exciton migration in photosynthetic reaction centers.

    PubMed

    Abramavicius, Darius; Mukamel, Shaul

    2010-08-14

    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.

  18. 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.

  19. Renin-angiotensin-aldosterone system inhibitors improve membrane stability and change gene-expression profiles in dystrophic skeletal muscles.

    PubMed

    Chadwick, Jessica A; Bhattacharya, Sayak; Lowe, Jeovanna; Weisleder, Noah; Rafael-Fortney, Jill A

    2017-02-01

    Angiotensin-converting enzyme inhibitors (ACEi) and mineralocorticoid receptor (MR) antagonists are FDA-approved drugs that inhibit the renin-angiotensin-aldosterone system (RAAS) and are used to treat heart failure. Combined treatment with the ACEi lisinopril and the nonspecific MR antagonist spironolactone surprisingly improves skeletal muscle, in addition to heart function and pathology in a Duchenne muscular dystrophy (DMD) mouse model. We recently demonstrated that MR is present in all limb and respiratory muscles and functions as a steroid hormone receptor in differentiated normal human skeletal muscle fibers. The goals of the current study were to begin to define cellular and molecular mechanisms mediating the skeletal muscle efficacy of RAAS inhibitor treatment. We also compared molecular changes resulting from RAAS inhibition with those resulting from the current DMD standard-of-care glucocorticoid treatment. Direct assessment of muscle membrane integrity demonstrated improvement in dystrophic mice treated with lisinopril and spironolactone compared with untreated mice. Short-term treatments of dystrophic mice with specific and nonspecific MR antagonists combined with lisinopril led to overlapping gene-expression profiles with beneficial regulation of metabolic processes and decreased inflammatory gene expression. Glucocorticoids increased apoptotic, proteolytic, and chemokine gene expression that was not changed by RAAS inhibitors in dystrophic mice. Microarray data identified potential genes that may underlie RAAS inhibitor treatment efficacy and the side effects of glucocorticoids. Direct effects of RAAS inhibitors on membrane integrity also contribute to improved pathology of dystrophic muscles. Together, these data will inform clinical development of MR antagonists for treating skeletal muscles in DMD.

  20. Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression

    PubMed Central

    Hanson, Michael A.; Brooun, Alexei; Baker, Kent A.; Jaakola, Veli-Pekka; Roth, Chris; Chien, Ellen; Alexandrov, Alexander; Velasquez, Jeffrey; Davis, Leila; Griffith, Mark; Moy, Kin; Ganser-Pornillos, Barbie; Kuhn, Peter; Ellis, Sam; Yeager, Mark; Stevens, Raymond C.

    2007-01-01

    Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple construct expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and coupled with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human β2-adrenergic receptor (β2AR) and gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of fourteen β2AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild type β2AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecyl maltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell micro-expression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants. PMID:17723307

  1. 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.

  2. 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.

  3. Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria

    PubMed Central

    Nevo, Reinat; Charuvi, Dana; Shimoni, Eyal; Schwarz, Rakefet; Kaplan, Aaron; Ohad, Itzhak; Reich, Ziv

    2007-01-01

    Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher-plant chloroplasts, allowing water-soluble and lipid-soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane-bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes. PMID:17304210

  4. Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria.

    PubMed

    Nevo, Reinat; Charuvi, Dana; Shimoni, Eyal; Schwarz, Rakefet; Kaplan, Aaron; Ohad, Itzhak; Reich, Ziv

    2007-03-07

    Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher-plant chloroplasts, allowing water-soluble and lipid-soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane-bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes.

  5. Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression.

    PubMed

    Hanson, Michael A; Brooun, Alexei; Baker, Kent A; Jaakola, Veli-Pekka; Roth, Chris; Chien, Ellen Y T; Alexandrov, Alexander; Velasquez, Jeffrey; Davis, Leila; Griffith, Mark; Moy, Kin; Ganser-Pornillos, Barbie K; Hua, Yuanzi; Kuhn, Peter; Ellis, Sam; Yeager, Mark; Stevens, Raymond C

    2007-11-01

    Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.

  6. 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

  7. Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

    PubMed Central

    Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Sun, Guochang

    2012-01-01

    Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium. PMID:23166741

  8. Growth temperature alters Salmonella Enteritidis heat/acid resistance, membrane lipid composition and stress/virulence related gene expression.

    PubMed

    Yang, Yishan; Khoo, Wei Jie; Zheng, Qianwang; Chung, Hyun-Jung; Yuk, Hyun-Gyun

    2014-02-17

    The influence of growth temperature (10, 25, 37, and 42 °C) on the survival of Salmonella Enteritidis in simulated gastric fluid (SGF; pH=2.0) and during heat treatment (54, 56, 58, and 60 °C), on the membrane fatty acid composition, as well as on stress-/virulence-related gene expression was studied. Cells incubated at temperatures lower or higher than 37 °C did not increase their acid resistance, with the maximum D-value of 3.07 min in cells grown at 37 °C; while those incubated at higher temperature increased their heat resistance, with the maximum D60 °C-values of 1.4 min in cells grown at 42 °C. A decrease in the ratio of unsaturated to saturated fatty acids was observed as the growth temperature increased. Compared to the control cells grown at 37 °C, the expression of rpoS was 16.5- and 14.4-fold higher in cells cultivated at 10 and 25 °C, respectively; while the expression of rpoH was 2.9-fold higher in those cultivated at 42 °C. The increased expression of stress response gene rpoH and the decreased ratio of unsaturated to saturated fatty acids correlated with the greater heat resistance of bacteria grown at 42 °C; while the decreased expression of stress response gene rpoS at 42 °C might contribute to the decrease in acid resistance. Virulence related genes-spvR, hilA, avrA-were induced in cells cultivated at 42 °C, except sefA which was induced in the control cells. This study indicates that environmental temperature may affect the virulence potential of S. Enteritidis, thus temperature should be well controlled during food storage.

  9. Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells.

    PubMed

    Tschantz, William R; Pfeifer, Nathan D; Meade, Caryl Lane; Wang, Leyu; Lanzetti, Anthony; Kamath, Ajith V; Berlioz-Seux, Francoise; Hashim, Muhammed F

    2008-02-01

    OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a K(m) of 5.9+/-0.76 microM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS-PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS-PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387 cm(-1) M(-1). Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.

  10. Novel B cell population producing functional IgG in the absence of membrane IgM expression.

    PubMed

    Orinska, Zane; Osiak, Anna; Löhler, Jürgen; Bulanova, Elena; Budagian, Vadim; Horak, Ivan; Bulfone-Paus, Silvia

    2002-12-01

    Surface expression of IgM is a characteristic feature of the development of most B cells. Only pre-B cells bearing functional IgM heavy chains mu chains) are selected for clonal expansion and differentiation. Cells lacking mu chains are normally eliminated. muMT mice carrying a deletion of the first exon coding for the transmembrane domain of the immunoglobulin mu chain gene were described as mice deficient for mature B cells, plasma cells and immunoglobulins in serum. In this study, we describe in muMT/BALB/c mice the presence of a novel B cell population, producing IgG, IgA and IgE in the absence of IgM membrane expression. Moreover, this small population of B cells is able to recognize antigens and to differentiate into plasma cells. These "non-conventional" mu(- / -) B cells produce functional immunoglobulins after immunization, undergo germinal center reactions, and maintain B cell memory. Our findings support the concept, that a small percentage of mu -non-expressing pre-B cells can escape elimination, switch to downstream immunoglobulin heavy chains and respond to antigens. It remains an open question how the reactivity of these B cells is regulated and in which extent such B cells play a role in physiological and pathological processes such as autoantibody production and autoimmunity.

  11. Neonatal, urogenital isolates of biotype 4 nontypeable Haemophilus influenzae express a variant P6 outer membrane protein molecule.

    PubMed Central

    Murphy, T F; Kirkham, C; Sikkema, D J

    1992-01-01

    The P6 outer membrane protein is a highly conserved molecule which is present on the surface of all strains of Haemophilus influenzae. Sixty strains of nontypeable H. influenzae which caused invasive disease or colonized the female urogenital tract were studied with monoclonal antibodies 7F3 and 4G4, which recognize different surface-exposed epitopes on the P6 molecule. All 60 strains expressed the epitope recognized by 4G4, whereas 47 of 60 strains expressed the epitope recognized by antibody 7F3. The 7F3-nonreactive strains were all biotype 4 and were recovered from the blood of neonates or postpartum women or from the female urogenital tract. The P6 genes from two 7F3-nonreactive strains were cloned, and the nucleotide sequences were determined. Analysis of amino acid sequences, immunoassays with synthetic peptides, and site-directed mutation of the P6 gene indicate that the epitope recognized by antibody 7F3 is conformational and that the sequence Asp-Ile-Thr is critical in maintaining the conformation of the epitope. We conclude that the unusually virulent clone family of biotype 4 strains of nontypeable H. influenzae express a variant P6 molecule which has an alteration in a highly conserved surface-exposed epitope. Images PMID:1373403

  12. Expression of multiple membrane-associated phospholipase A1 beta transcript variants and lysophosphatidic acid receptors in Ewing tumor cells.

    PubMed

    Schmiedel, Benjamin Joachim; Hutter, Christoph; Hesse, Manuela; Staege, Martin Sebastian

    2011-10-01

    The prognosis for patients with advanced stages of Ewing family tumors (EFT) is very poor. EFT express high levels of phosphatidic acid specific membrane-associated phospholipase A1 beta (lipase I, LIPI). LIPI is a cancer/testis antigen and the high tumor specificity suggests that LIPI might be an attractive target for new diagnostic and/or therapeutic developments. By using reverse transcriptase-polymerase chain reaction (RT-PCR), we observed simultaneous presence of multiple LIPI transcript variants in EFT. We cloned and sequenced these transcript variants from EFT cell lines. Sequence analysis indicated that all transcript variants were derived by alternative splicing. Homology modeling of corresponding protein structures suggested that different transcript variants differ in their regulatory lid domains. In addition, expression of receptors for lysophosphatidic acid (LPA) was analyzed in a panel of EFT cell lines by RT-PCR. We observed that EFT cell lines expressed high levels of LPA receptors. Different LIPI transcript variants present in EFT might be involved in the pathogenesis of EFT by signaling via these LPA receptors.

  13. 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.

  14. 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.

  15. Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

    PubMed

    Rudtanatip, Tawut; Withyachumnarnkul, Boonsirm; Wongprasert, Kanokpan

    2015-11-01

    Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and β-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway.

  16. Correlation between Resistance of Pseudomonas aeruginosa to Quaternary Ammonium Compounds and Expression of Outer Membrane Protein OprR

    PubMed Central

    Tabata, Atsushi; Nagamune, Hideaki; Maeda, Takuya; Murakami, Keiji; Miyake, Yoichiro; Kourai, Hiroki

    2003-01-01

    The adaptation mechanism of Pseudomonas aeruginosa ATCC 10145 to quaternary ammonium compounds (QACs) was investigated. A P. aeruginosa strain with adapted resistance to QACs was developed by a standard broth dilution method. It was revealed that P. aeruginosa exhibited remarkable resistance to N-dodecylpyridinium iodide (P-12), whose structure is similar to that of a common disinfectant, cetylpyridinium chloride. Adapted resistance to benzalkonium chloride (BAC), which is commonly used as a disinfectant, was also observed in P. aeruginosa. Moreover, the P-12-resistant strain exhibited cross-resistance to BAC. Analysis of the outer membrane protein of the P-12-resistant strain by two-dimensional polyacrylamide gel electrophoresis showed a significant increase in the level of expression of a protein (named OprR) whose molecular mass was approximately 26 kDa. The actual function of OprR is not yet clear; however, OprR was expected to be an outer membrane-associated protein with homology to lipoproteins of other bacterial species, according to a search of the National Center for Biotechnology Information website with the BLAST program by use of the N-terminal sequence of OprR. A correlation between the level of expression of OprR and the level of resistance of P. aeruginosa to QACs was observed by using a PA2800 gene knockout mutant derived from the P-12-resistant strain. The knockout mutant recovered susceptibility not only to P-12 but also to BAC. These results suggested that OprR significantly participated in the adaptation of P. aeruginosa to QACs, such as P-12 and BAC. PMID:12821452

  17. 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.

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

    DOE PAGES

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; ...

    2016-01-21

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. 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

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

    SciTech Connect

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Urban, Volker S.; 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-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. 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.

  20. 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.

  1. In vitro expressed GPCR inserted in polymersome membranes for ligand-binding studies.

    PubMed

    May, Sylvia; Andreasson-Ochsner, Mirjam; Fu, Zhikang; Low, Ying Xiu; Tan, Darren; de Hoog, Hans-Peter M; Ritz, Sandra; Nallani, Madhavan; Sinner, Eva-Kathrin

    2013-01-07

    The dopamine receptor D2 (DRD2), a G-protein coupled receptor is expressed into PBd(22)-PEO(13) and PMOXA(20)-PDMS(54)-PMOXA(20) block copolymer vesicles. The conformational integrity of the receptor is confirmed by antibody- and ligand-binding assays. Replacement of bound dopamine is demonstrated on surface-immobilized polymersomes, thus making this a promising platform for drug screening.

  2. 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.

  3. 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

  4. Role of membrane potential and expression of endothelial factors in restenosis after angioplasty in SHR.

    PubMed

    Dina, Janaina P; Feres, Teresa; Paiva, Antonio C M; Paiva, Therezinha B

    2004-01-01

    We examined the roles played by impaired K+ channels, diminished nitric oxide (NO) production, endothelin release, and smooth muscle membrane potential in the increased restenosis observed in spontaneously hypertensive rat (SHR) carotid arteries after angioplasty. The SHR carotid was found to be less polarized than that of normotensive Wistar rats (NWR), and it was further depolarized by the alpha2 agonist UK 14,304. This response was blocked by iberiotoxin, indicating that calcium-dependent K+ channels operate normally in the SHR carotid. Acetylcholine caused a hyperpolarization that was significantly smaller in SHR than in NWR carotids, indicating a deficient release of NO in the SHR. After angioplasty, SHR and NWR vessels were depolarized, returning to baseline after 10 days. In the SHR but not in the NWR the contralateral carotid was also depolarized, and this was prevented by the endothelin A/B receptor antagonist bosentan. After angioplasty, endothelin-1 plasma levels increased in both SHR and NWR, but the increase was significantly more prolonged in SHR. We found that the more pronounced restenosis observed in the SHR carotid after angioplasty is not due to impairment of calcium-dependent K+ channels but is related to the relatively depolarized vascular smooth muscles, involving endothelin release caused by reduced NO levels in that strain.

  5. The significance of the increased expression of phosphorylated MeCP2 in the membranes from patients with proliferative diabetic retinopathy

    PubMed Central

    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

  6. Drought, salt and wounding stress induce the expression of the plasma membrane intrinsic protein 1 gene in poplar (Populus alba×P. tremula var. glandulosa).

    PubMed

    Bae, Eun-Kyung; Lee, Hyoshin; Lee, Jae-Soon; Noh, Eun-Woon

    2011-09-01

    Water uptake across cell membranes is a principal requirement for plant growth at both the cellular and whole-plant levels; water movement through plant membranes is regulated by aquaporins (AQPs) or major intrinsic proteins (MIPs). We examined the expression characteristics of the poplar plasma membrane intrinsic protein 1 gene (PatPIP1), a type of MIP, which was isolated from a suspension cell cDNA library of Populus alba×P. tremula var. glandulosa. Examination of protoplasts expressing the p35S-PatPIP1::sGFP fusion protein revealed that the protein was localized in the plasma membrane. Northern blot analysis revealed that the gene was strongly expressed in poplar roots and leaves. Gene expression was inducible by abiotic factors including drought, salinity, cold temperatures and wounding, and also by plant hormones including gibberellic acid, jasmonic acid and salicylic acid. Since we found that the PatPIP1 gene was strongly expressed in response to mannitol, NaCl, jasmonic acid and wounding, we propose that PatPIP1 plays an essential role in the defense of plants against water stress.

  7. 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

  8. Metformin Transport by a Newly Cloned Proton-Stimulated Organic Cation Transporter (Plasma Membrane Monoamine Transporter) Expressed in Human Intestine

    PubMed Central

    Zhou, Mingyan; Xia, Li; Wang, Joanne

    2009-01-01

    Metformin is a widely used oral antihyperglycemic drug for the treatment of type II diabetes mellitus. The intestinal absorption of metformin is dose-dependent and involves an active, saturable uptake process. Metformin has been shown to be transported by the human organic cation transporters 1 and 2 (hOCT1–2). We recently cloned and characterized a novel proton-activated organic cation transporter, plasma membrane monoamine transporter (PMAT). We previously showed that PMAT transports many classic organic cations (e.g., monoamine neurotransmitters, 1-methyl-4-phenylpyridinium) in a pH-dependent manner and its mRNA is expressed in multiple human tissues. The goal of this study is to investigate whether metformin is a substrate of PMAT and whether PMAT plays a role in the intestinal uptake of metformin. Using Madin-Darby canine kidney cells stably expressing human PMAT, we showed that metformin is avidly transported by PMAT, with an apparent affinity (Km = 1.32 mM) comparable to those reported for hOCT1–2. Interestingly, the concentration-velocity profile of PMAT-mediated metformin uptake is sigmoidal, with a Hill coefficient of 2.64. PMAT-mediated metformin transport is greatly stimulated by acidic pH, with the uptake rate being ~4-fold higher at pH 6.6 than at pH 7.4. Using a polyclonal antibody against PMAT, we showed that the PMAT protein (58 kDa) was expressed in human small intestine and concentrated on the tips of the mucosal epithelial layer. Taken together, our results suggest that PMAT transports metformin, is expressed in human intestine, and may play a role in the intestinal absorption of metformin and possibly other cationic drugs. PMID:17600084

  9. 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

  10. Carotenoid cation formation and the regulation of photosynthetic light harvesting.

    PubMed

    Holt, Nancy E; Zigmantas, Donatas; Valkunas, Leonas; Li, Xiao-Ping; Niyogi, Krishna K; Fleming, Graham R

    2005-01-21

    Photosynthetic light harvesting in excess light is regulated by a process known as feedback deexcitation. Femtosecond transient absorption measurements on thylakoid membranes show selective formation of a carotenoid radical cation upon excitation of chlorophyll under conditions of maximum, steady-state feedback deexcitation. Studies on transgenic Arabidopsis thaliana plants confirmed that this carotenoid radical cation formation is correlated with feedback deexcitation and requires the presence of zeaxanthin, the specific carotenoid synthesized during high light exposure. These results indicate that energy transfer from chlorophyll molecules to a chlorophyllzeaxanthin heterodimer, which then undergoes charge separation, is the mechanism for excess energy dissipation during feedback deexcitation.

  11. Triazine herbicide resistance in the photosynthetic bacterium Rhodopseudomonas sphaeroides

    PubMed Central

    Brown, Alfred E.; Gilbert, Carl W.; Guy, Rachel; Arntzen, Charles J.

    1984-01-01

    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 QB protein of chloroplast membranes. Images PMID:16593520

  12. Expression Patterns of MicroRNAs in the Chorioamniotic Membranes: a Role for MicroRNAs in Human Pregnancy and Parturition

    PubMed Central

    Montenegro, Daniel; Romero, Roberto; Kim, Sung-Su; Tarca, Adi L; Draghici, Sorin; Kusanovic, Juan Pedro; Kim, Jung-Sun; Lee, Deug-Chan; Erez, Offer; Gotsch, Francesca; Hassan, Sonia S.; Kim, Chong Jai

    2014-01-01

    MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of gene expression during development. This study was performed to determine gestational age-dependent changes in miRNA expression in the chorioamniotic membranes and to assess the significance of miRNAs in human pregnancy and parturition. The expression profile of 455 miRNAs was compared between patients at term without labor (TNL: n=10), in labor (TL: n=10), and preterm labor (PTL: n=10) using microarrays. A total of 39 miRNAs were differentially expressed between term and preterm cases, of which 31 (79.5%) were down-regulated at term. Expression of 10 miRNAs, including miR-338, differentially expressed between PTL and TL groups was decreased at term. Computational analyses using miRBase Targets have identified PLA2G4B, a phospholipase implicated in parturition, as a putative target of miR-338. Inhibition of endogenous miR-338 with anti-miR-338 increased the mRNA and protein expression of PLA2G4B in decidual cells. Luciferase assay with reporter constructs confirmed that the suppression of PLA2G4B occurs through binding of miR-338 to the 3’UTR of PLA2G4B. Interestingly, the expression of Dicer, a key miRNA-processing enzyme, was markedly decreased at term, particularly with labor in the chorioamniotic membranes. Collectively, the novel findings reported herein strongly suggest that post-transcriptional regulation of genes by miRNAs, coupled with the changes of miRNA processing machinery in the chorioamniotic membranes, plays a role in pregnancy and parturition. Furthermore, the expression level of Dicer in the chorioamniotic membranes dichotomizes pathological preterm labor and physiological spontaneous labor at term. PMID:18991333

  13. 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

  14. 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.

  15. Spatial and temporal expression profiles suggest the involvement of gelatinase A and membrane type 1 matrix metalloproteinase in amphibian metamorphosis.

    PubMed

    Hasebe, Takashi; Hartman, Rebecca; Matsuda, Hiroki; Shi, Yun-Bo

    2006-04-01

    The matrix metalloproteinases (MMPs) are a family of proteases capable of degrading various components of the extracellular matrix (ECM). Among them, the membrane type MMP-1 (MT1-MMP) has been shown to participate in the activation of MMP gelatinase A (GelA), suggesting that they may function together in development and pathogenesis. Here, we have investigated the spatiotemporal expression profiles of Xenopus laevis MT1-MMP and GelA genes during thyroid-hormone-dependent metamorphosis. We have focused our studies on two organs: (1) the intestine, which undergoes first the degeneration of the tadpole epithelium through apoptosis and then the development of adult epithelium and other tissues, and (2) the tail, which completely resorbs through programmed cell death. We show that both MT1-MMP and GelA are upregulated in the intestine and tail when both organs undergo metamorphosis. Within the organs, MT1-MMP and GelA are coexpressed in the connective tissues during both natural and thyroid-hormone-induced metamorphosis. In addition, MT1-MMP (but not GelA) is also expressed in the longitudinal muscle cells of the metamorphosing intestine. These results suggest that MT1-MMP and GelA function together in the ECM degradation or remodeling associated with metamorphosis and that MT1-MMP has additional GelA-independent roles in the development of adult longitudinal muscle in the intestine.

  16. Expression of nestin, mesothelin and epithelial membrane antigen (EMA) in developing and adult human meninges and meningiomas.

    PubMed

    Petricevic, Josko; Forempoher, Gea; Ostojic, Ljerka; Mardesic-Brakus, Snjezana; Andjelinovic, Simun; Vukojevic, Katarina; Saraga-Babic, Mirna

    2011-11-01

    The spatial and temporal pattern of appearance of nestin, epithelial membrane antigen (EMA) and mesothelin proteins was immunohistochemically determined in the cells of normal developing and adult human meninges and meningiomas. Human meninges developed as two mesenchymal condensations in the head region. The simple squamous epithelium on the surface of leptomeninges developed during mesenchymal to epithelial transformation. Nestin appeared for the first time in week 7, EMA in week 8, while mesothelin appeared in week 22 of development. In the late fetal period and after birth, nestin expression decreased, whereas expression of EMA and mesothelin increased. EMA appeared in all surface epithelial cells and nodules, while mesothelin was found only in some of them. In adult meninges, all three proteins were predominantly localized in the surface epithelium and meningeal nodules. In meningothelial meningiomas (WHO grade I), EMA was detected in all tumor cells except in the endothelial cells, mesothelin characterized nests of tumor cells, while nestin was found predominantly in the walls of blood vessels. The distribution pattern of those proteins in normal meningeal and tumor cells indicates that nestin might characterize immature cells, while EMA and mesothelin appeared in maturing epithelial cells. Neoplastic transformation of these specific cell lineages contributes to the cell population in meningiomas.

  17. Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer

    PubMed Central

    Peng, Zheng-Hong; Sima, Monika; Salama, Mohamed E.; Kopečková, Pavla; Kopeček, Jindřich

    2015-01-01

    Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (–GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer – DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates’ in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice. PMID:24160903

  18. 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.

  19. 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.

  20. Expression of DNAJB12 or DNAJB14 causes coordinate invasion of the nucleus by membranes associated with a novel nuclear pore structure.

    PubMed

    Goodwin, Edward C; Motamedi, Nasim; Lipovsky, Alex; Fernández-Busnadiego, Rubén; DiMaio, Daniel

    2014-01-01

    DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity.

  1. Protection of the photosynthetic apparatus against dehydration stress in the resurrection plant Craterostigma pumilum.

    PubMed

    Zia, Ahmad; Walker, Berkley J; Oung, Hui Min Olivia; Charuvi, Dana; Jahns, Peter; Cousins, Asaph B; Farrant, Jill M; Reich, Ziv; Kirchhoff, Helmut

    2016-09-01

    The group of homoiochlorophyllous resurrection plants evolved the unique capability to survive severe drought stress without dismantling the photosynthetic machinery. This implies that they developed efficient strategies to protect the leaves from reactive oxygen species (ROS) generated by photosynthetic side reactions. These strategies, however, are poorly understood. Here, we performed a detailed study of the photosynthetic machinery in the homoiochlorophyllous resurrection plant Craterostigma pumilum during dehydration and upon recovery from desiccation. During dehydration and rehydration, C. pumilum deactivates and activates partial components of the photosynthetic machinery in a specific order, allowing for coordinated shutdown and subsequent reinstatement of photosynthesis. Early responses to dehydration are the closure of stomata and activation of electron transfer to oxygen accompanied by inactivation of the cytochrome b6 f complex leading to attenuation of the photosynthetic linear electron flux (LEF). The decline in LEF is paralleled by a gradual increase in cyclic electron transport to maintain ATP production. At low water contents, inactivation and supramolecular reorganization of photosystem II becomes apparent, accompanied by functional detachment of light-harvesting complexes and interrupted access to plastoquinone. This well-ordered sequence of alterations in the photosynthetic thylakoid membranes helps prepare the plant for the desiccated state and minimize ROS production.

  2. Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

    PubMed

    Fogeron, Marie-Laure; Badillo, Aurélie; Jirasko, Vlastimil; Gouttenoire, Jérôme; Paul, David; Lancien, Loick; Moradpour, Darius; Bartenschlager, Ralf; Meier, Beat H; Penin, François; Böckmann, Anja

    2015-01-01

    Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

  3. 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

  4. Initial characterization of the photosynthetic apparatus of "Candidatus Chlorothrix halophila," a filamentous, anoxygenic photoautotroph.

    PubMed

    van de Meene, Allison M L; Le Olson, Tien; Collins, Aaron M; Blankenship, Robert E

    2007-06-01

    "Candidatus Chlorothrix halophila" is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181:17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of "Ca. Chlorothrix halophila," that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an approximately 6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of "Ca. Chlorothrix halophila" revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus.

  5. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane.

    PubMed

    Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh

    2016-12-01

    Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.

  6. A putative role for the plasma membrane potential in the control of the expression of the gene encoding the tomato high-affinity potassium transporter HAK5.

    PubMed

    Nieves-Cordones, Manuel; Miller, Anthony J; Alemán, Fernando; Martínez, Vicente; Rubio, Francisco

    2008-12-01

    A chimeric CaHAK1-LeHAK5 transporter with only 15 amino acids of CaHAK1 in the N-terminus mediates high-affinity K(+) uptake in yeast cells. Kinetic and expression analyses strongly suggest that LeHAK5 mediates a significant proportion of the high-affinity K(+) uptake shown by K(+)-starved tomato (Solanum lycopersicum) plants. The development of high-affinity K(+) uptake, putatively mediated by LeHAK5, was correlated with increased LeHAK5 mRNA levels and a more negative electrical potential difference across the plasma membrane of root epidermal and cortical cells. However, this increase in high-affinity K(+) uptake was not correlated with the root K(+) content. Thus, (i) growth conditions that result in a hyperpolarized root plasma membrane potential, such as K(+) starvation or growth in the presence of NH(4) (+), but which do not decrease the K(+) content, lead to increased LeHAK5 expression; (ii) the presence of NaCl in the growth solution, which prevents the hyperpolarization induced by K(+) starvation, also prevents LeHAK5 expression. Moreover, once the gene is induced, depolarization of the plasma membrane potential then produces a decrease in the LeHAK5 mRNA. On the basis of these results, we propose that the plant membrane electrical potential plays a role in the regulation of the expression of this gene encoding a high-affinity K(+) transporter.

  7. Stoichiometry and kinetics of mercury uptake by photosynthetic bacteria.

    PubMed

    Kis, Mariann; Sipka, Gábor; Maróti, Péter

    2017-03-04

    Mercury adsorption on the cell surface and intracellular uptake by bacteria represent the key first step in the production and accumulation of highly toxic mercury in living organisms. In this work, the biophysical characteristics of mercury bioaccumulation are studied in intact cells of photosynthetic bacteria by use of analytical (dithizone) assay and physiological photosynthetic markers (pigment content, fluorescence induction, and membrane potential) to determine the amount of mercury ions bound to the cell surface and taken up by the cell. It is shown that the Hg(II) uptake mechanism (1) has two kinetically distinguishable components, (2) includes co-opted influx through heavy metal transporters since the slow component is inhibited by Ca(2+) channel blockers, (3) shows complex pH dependence demonstrating the competition of ligand binding of Hg(II) ions with H(+) ions (low pH) and high tendency of complex formation of Hg(II) with hydroxyl ions (high pH), and (4) is not a passive but an energy-dependent process as evidenced by light activation and inhibition by protonophore. Photosynthetic bacteria can accumulate Hg(II) in amounts much (about 10(5)) greater than their own masses by well-defined strong and weak binding sites with equilibrium binding constants in the range of 1 (μM)(-1) and 1 (mM)(-1), respectively. The strong binding sites are attributed to sulfhydryl groups as the uptake is blocked by use of sulfhydryl modifying agents and their number is much (two orders of magnitude) smaller than the number of weak binding sites. Biofilms developed by some bacteria (e.g., Rvx. gelatinosus) increase the mercury binding capacity further by a factor of about five. Photosynthetic bacteria in the light act as a sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury-contaminated aqueous cultures.

  8. Thirsty business: cell, region, and membrane specificity of aquaporins in the testis, efferent ducts, and epididymis and factors regulating their expression.

    PubMed

    Hermo, Louis; Smith, Charles E

    2011-01-01

    Water content within the male reproductive tract is stringently regulated in order to promote sperm differentiation and maturation. Aquaporins (AQP) are a family of integral membrane proteins allowing the transcellular transport of water, gases, urea, glycerol, and ions. Past studies from our lab have revealed the following. In the testis, Sertoli cells express AQP 8, whereas germ cells express AQP 7. In the efferent ducts (ED), AQP 1, 9, and 10 localize to microvilli of nonciliated cells, in addition to a basolateral staining for AQP 1, whereas AQP 1 and 10 localize to ciliated cells. AQP 7 and 11 are expressed in the ED epithelium of young but not adult rats, suggesting suppression of translation as rats age. In the adult epididymis, AQP 1 appears in endothelial cells of vascular channels and myoid cells, whereas AQP 3 delineates basal cells. In principal cells, AQP 9 and 11 appear on microvilli, whereas AQP 7 localizes to lateral then to basal plasma membranes in a region-specific manner; AQP 7 also associates with myoid cells. AQP 5 is expressed in corpus and cauda regions. Additionally, several AQPs are expressed by some but not all basal (AQP 7, 11), clear (AQP 7, 9), and halo (AQP 7, 11) cells. Regulation studies reveal a role for estrogen, androgens, and lumicrine factors. These findings indicate unique associations of AQPs with specific membrane domains in a cell type- and region-specific manner within the EDs and epididymis, as well as complex regulation patterns of expression.

  9. Enhancement of AMPA currents and GluR1 membrane expression through PKA-coupled adenosine A(2A) receptors.

    PubMed

    Dias, Raquel B; Ribeiro, Joaquim A; Sebastião, Ana M

    2012-02-01

    Phosphorylation of glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by Protein Kinase A (PKA) is known to regulate AMPA receptor (AMPAR) trafficking and stabilization at the postsynaptic membrane, which in turn is one of the key mechanisms by which synaptic transmission and plasticity are tuned. However, not much is known as to how Gs-coupled receptors contribute to endogenous PKA-mediated regulation of AMPA receptor function. Here we report that activation of the excitatory A(2A) adenosine receptor by 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, 1-30 nM) facilitates AMPA-evoked currents in CA1 pyramidal neurons, by a mechanism dependent on PKA activation, but not on protein synthesis. This modulation of AMPA currents was mimicked by forskolin (1 μM) and did not occur in stratum radiatum interneurons. Superfusion of the A(2A) receptor agonist also caused an increase in the amplitude of miniature excitatory postsynaptic currents (mEPSCs), as well as in the membrane levels of GluR1 subunits phosphorylated at the PKA site (Ser845). The impact of this increase on GluR1-containing AMPA receptor expression was evidenced by the potentiation of LTP at the CA3-CA1 synapse that followed brief activation of A(2A) receptors. We thus propose that in conditions of increased adenosine availability, A(2A) receptor activation is responsible for setting part of the endogenous GluR1 Ser-845 phosphorylation tonus and hence, the availability of the GluR1-containing AMPA receptor extrasynaptic pool for synaptic insertion and reinforcement of synaptic strength.

  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 and function of thyroid hormone transporters in the microvillous plasma membrane of human term placental syncytiotrophoblast.

    PubMed

    Loubière, L S; Vasilopoulou, E; Glazier, J D; Taylor, P M; Franklyn, J A; Kilby, M D; Chan, Shiao Y

    2012-12-01

    The transplacental passage of thyroid hormones (THs) from mother to fetus in humans has been deduced from observational clinical studies and is important for normal fetoplacental development. To investigate the transporters that regulate TH uptake by syncytiotrophoblast (the primary barrier to maternal-fetal exchange, which lies in direct contact with maternal blood), we isolated the microvillous plasma membrane (MVM) of human term syncytiotrophoblasts. We have demonstrated that MVM vesicles express plasma membrane TH transporter proteins, including system-L (L-type amino acid transporter 1 and CD98), monocarboxylate transporters (MCTs) 8 and 10, organic anion-transporting polypeptides 1A2 and 4A1. We provide the first definitive evidence that the human syncytiotrophoblast MVM is capable of rapid, saturable T(4) and T(3) uptake at similar rates and in a Na(+)-independent manner. These two major forms of THs could not significantly inhibit each others' uptake, suggesting that each is mediated by largely different transporters. No single transporter was noted to play a dominant role in either T(4) or T(3) uptake. Using combinations of transporter inhibitors that had an additive effect on TH uptake, we provide evidence that 67% of saturable T(4) uptake is facilitated by system-L and MCT10 with a minor role played by organic anion-transporting polypeptides, whereas 87% of saturable T(3) uptake is mediated by MCT8 and MCT10. Our data demonstrate that syncytiotrophoblast may control the quantity and forms of THs taken up by the human placenta. Thus, syncytiotrophoblast could be critical in regulating transplacental TH supply from the mother to the fetus.

  12. Construction and Structural Analysis of Tethered Lipid Bilayer Containing Photosynthetic Antenna Proteins for Functional Analysis

    SciTech Connect

    Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu; Sugiura, Ryuta; Sasaki, Nobuaki; Misawa, Nobuo; Tero, Ryugo; Urisu, Tsuneo; Gardiner, Alastair T.; Cogdell, Richard J.; Hashimoto, Hideki; Nango, Mamoru

    2011-07-11

    The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAY), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

  13. Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes

    PubMed Central

    Ross, GD; Lambris, JD

    1982-01-01

    Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab’)(2) anti-CR(1) (C3b receptor) or F(ab’)(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab’)(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab’)(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation

  14. IM30 triggers membrane fusion in cyanobacteria and chloroplasts.

    PubMed

    Hennig, Raoul; Heidrich, Jennifer; Saur, Michael; Schmüser, Lars; Roeters, Steven J; Hellmann, Nadja; Woutersen, Sander; Bonn, Mischa; Weidner, Tobias; Markl, Jürgen; Schneider, Dirk

    2015-05-08

    The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane fusion events are essential for the formation of thylakoid membranes, proteins involved in membrane fusion have yet to be identified in photosynthetic cells or organelles. Here we show that IM30, a conserved chloroplast and cyanobacterial protein of approximately 30 kDa binds as an oligomeric ring in a well-defined geometry specifically to membranes containing anionic lipids. Triggered by Mg(2+), membrane binding causes destabilization and eventually results in membrane fusion. We propose that IM30 establishes contacts between internal membrane sites and promotes fusion to enable regulated exchange of proteins and/or lipids in cyanobacteria and chloroplasts.

  15. Protein import into the photosynthetic organelles of Paulinella chromatophora and its implications for primary plastid endosymbiosis.

    PubMed

    Mackiewicz, Paweł; Bodył, Andrzej; Gagat, Przemysław

    2012-12-01

    The rhizarian amoeba Paulinella chromatophora harbors two photosynthetically active organelles of cyanobacterial origin that have been acquired independently of classic primary plastids. Because their acquisition did take place relatively recently, they are expected to provide new insight into the ancient cyanobacterial primary endosymbiosis. During the process of Paulinella endosymbiont-to-organelle transformation, more than 30 genes have been transferred from the organelle to the host nuclear genome via endosymbiotic gene transfer (EGT). The article discusses step-by-step protein import of EGT-derived proteins into Paulinella photosynthetic organelles with the emphasis on the nature of their targeting signals and the final passage of proteins through the inner organelle membrane. The latter most probably involves a simplified Tic translocon composed of Tic21- and Tic32-like proteins as well as a Hsp70-based motor responsible for pulling of imported proteins into the organelle matrix. Our results indicate that although protein translocation across the inner membrane of Paulinella photosynthetic organelles seems to resemble the one in classic primary plastids, the transport through the outer membrane does not. The differences could result from distinct integration pathways of Paulinella photosynthetic organelles and primary plastids with their respective host cells.

  16. In vivo administration of extracellular cGMP normalizes TNF-α and membrane expression of AMPA receptors in hippocampus and spatial reference memory but not IL-1β, NMDA receptors in membrane and working memory in hyperammonemic rats.

    PubMed

    Cabrera-Pastor, Andrea; Hernandez-Rabaza, Vicente; Taoro-Gonzalez, Lucas; Balzano, Tiziano; Llansola, Marta; Felipo, Vicente

    2016-10-01

    Patients with hepatic encephalopathy (HE) show working memory and visuo-spatial orientation deficits. Hyperammonemia is a main contributor to cognitive impairment in HE. Hyperammonemic rats show impaired spatial learning and learning ability in the Y maze. Intracerebral administration of extracellular cGMP restores learning in the Y-maze. The underlying mechanisms remain unknown. It also remains unknown whether extracellular cGMP improves neuroinflammation or restores spatial learning in hyperammonemic rats and if it affects differently reference and working memory. The aims of this work were: Spatial working and reference memory were assessed using the radial and Morris water mazes and neuroinflammation by immunohistochemistry and Western blot. Membrane expression of NMDA and AMPA receptor subunits was analyzed using the BS3 crosslinker. Extracellular cGMP was administered intracerebrally using osmotic minipumps. Chronic hyperammonemia induces neuroinflammation in hippocampus, with astrocytes activation and increased IL-1β, which are associated with increased NMDA receptors membrane expression and impaired working memory. This process is not affected by extracellular cGMP. Hyperammonemia also activates microglia and increases TNF-α, alters membrane expression of AMPA receptor subunits (increased GluA1 and reduced GluA2) and impairs reference memory. All these changes are reversed by extracellular cGMP. These results show that extracellular cGMP modulates spatial reference memory but not working memory. This would be mediated by modulation of TNF-α levels and of membrane expression of GluA1 and GluA2 subunits of AMPA receptors.

  17. Functional Expression in Escherichia coli and Membrane Topology of Porin HopE, a Member of a Large Family of Conserved Proteins in Helicobacter pylori

    PubMed Central

    Bina, Jim; Bains, Manjeet; Hancock, Robert E. W.

    2000-01-01

    HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin. In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from E. coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H. pylori. A model of the membrane topology of HopE was constructed and indicated that this protein formed a β-barrel with 16 transmembrane amphipathic β-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane β-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane. PMID:10762234

  18. Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

    PubMed Central

    Monet, Michael; Birgy-Barelli, Eléonore; Léna, Isabelle; Counillon, Laurent

    2015-01-01

    Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection. PMID:25867523

  19. Isolation and characterization of chimeric human Fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow.

    PubMed

    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

    2014-01-08

    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

  20. 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.

  1. Evaluation of murine lymphocyte membrane potential, intracellular free calcium, and interleukin-2 receptor expression upon exposure to 1,1-dimethylhydrazine.

    PubMed

    Frazier, D E; Tarr, M J; Olsen, R G

    1992-06-01

    The effects of 1,1-dimethylhydrazine (UDMH) on several early events associated with lymphocyte activation were examined. The concentration of intracellular calcium ([Ca2+]i) and membrane potential of murine lymphocytes were found to be altered upon exposure to UDMH; [Ca2+]i was increased in murine thymocytes, while splenocytes exhibited membrane hyperpolarization. In addition, interleukin-2 receptor expression induced by in-vitro concanavalin A stimulation of murine splenocytes at 24 and 48 h in the presence of UDMH was not affected. UDMH may interfere with the ability of these two distinct lymphocyte populations to regulate normal ionic fluctuations, thus contributing to altered immune responsiveness.

  2. 1D4: a versatile epitope tag for the purification and characterization of expressed membrane and soluble proteins.

    PubMed

    Molday, Laurie L; Molday, Robert S

    2014-01-01

    Incorporation of short epitope tags into proteins for recognition by commercially available monoclonal or polyclonal antibodies has greatly facilitated the detection, characterization, localization, and purification of heterologously expressed proteins for structure-function studies. A number of tags have been developed, but many epitope-antibody combinations do not work effectively for all immunochemical techniques due to the nature of the tag and the specificity of the antibodies. A highly versatile, multipurpose epitope tag is the 9 amino acid C-terminal 1D4 peptide. This peptide tag together with the Rho1D4 monoclonal antibody can be used to detect proteins in complex mixtures by western blotting and ELISA assays, localize proteins in cells by immunofluorescence and immunoelectron microscopic labeling techniques, identify subunits and interacting proteins by co-immunoprecipitation, and purify functionally active proteins including membrane proteins by immunoaffinity chromatography. In this chapter we describe various immunochemical procedures which can be used for the detection, purification and localization of 1D4-tagged proteins for structure-function studies.

  3. Expression, purification and crystallization of a membrane-associated, catalytically active type I signal peptidase from Staphylococcus aureus.

    PubMed

    Ting, Yi Tian; Batot, Gaëlle; Baker, Edward N; Young, Paul G

    2015-01-01

    Staphylococcus aureus infections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets. S. aureus has a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, the spsB gene from S. aureus Newman strain was cloned and overexpressed in Escherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space group P2(1) and diffracted to 2.05 Å resolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases.

  4. 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.

  5. Increased expression of a phloem membrane protein encoded by NHL26 alters phloem export and sugar partitioning in Arabidopsis.

    PubMed

    Vilaine, Françoise; Kerchev, Pavel; Clément, Gilles; Batailler, Brigitte; Cayla, Thibaud; Bill, Laurence; Gissot, Lionel; Dinant, Sylvie

    2013-05-01

    The complex process of phloem sugar transport involves symplasmic and apoplasmic events. We characterized Arabidopsis thaliana lines ectopically expressing a phloem-specific gene encoding NDR1/HIN1-like26 (NHL26), a putative membrane protein. NHL26 overexpressor plants grew more slowly than wild-type plants, accumulated high levels of carbohydrates in mature leaves, and had a higher shoot biomass, contrasting with slower root growth and a lower seed yield. Similar effects were observed when NHL26 was overexpressed in companion cells, under the control of a companion cell-specific promoter. The soluble sugar content of the phloem sap and sink organs was lower than that in the wild type, providing evidence of a sugar export defect. This was confirmed in a phloem-export assay with the symplastic tracer carboxyfluorescein diacetate. Leaf sugar accumulation was accompanied by higher organic acid, amino acid, and protein contents, whereas analysis of the metabolite profile of phloem sap exudate revealed no change in amino acid or organic acid content, indicating a specific effect on sugar export. NHL26 was found to be located in the phloem plasmodesmata and the endoplasmic reticulum. These findings reveal that NHL26 accumulation affects either the permeability of plasmodesmata or sugar signaling in companion cells, with a specific effect on sugar export.

  6. Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150).

    PubMed Central

    Look, A T; Peiper, S C; Rebentisch, M B; Ashmun, R A; Roussel, M F; Lemons, R S; Le Beau, M M; Rubin, C M; Sherr, C J

    1986-01-01

    DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells. Images PMID:2428842

  7. 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

  8. The relationship between follicle development and progesterone receptor membrane component-1 (PGRMC1) expression in women undergoing in vitro fertilization

    PubMed Central

    Elassar, Alyaa; Liu, Xiufang; Scranton, Victoria; Wu, Carol A.; Peluso, John J.

    2012-01-01

    Objective To determine the relationship between Progesterone Receptor Membrane Component-1 (PGRMC1) expression and the outcome of in vitro fertilization treatment. Design A prospective study in which PGRMC1 mRNA levels, methylation status of the Pgrmc1 promoter, and the presence of point mutations within Pgrmc1 were obtained from granulosa/luteal cells of women undergoing controlled ovarian hyperstimulation (COH). Setting Fertility center/Basic science laboratory Patients Eighty-five IVF patients and 10 women, who were undergoing COH for the purpose of oocyte donation, were included in this study. Interventions None. Main Outcome Measures PGRMC1 measurements were correlated with clinical outcomes, such as number of follicles, number of retrieved oocytes and ongoing pregnancy rates. Results PGRMC1 mRNA levels within granulosa/luteal cells of 18% of IVF patients were > 2.25 fold higher than those of oocyte donors. Individuals with elevated PGRMC1 mRNA levels had 30% fewer large follicles and fewer oocytes retrieved. The elevated PGRMC1 mRNA levels were associated with an increase in the methylation of Pgrmc1 promoter. Conclusion In patients with elevated PGRMC1 mRNA levels, gonadotropin-induced follicle development is attenuated, although sufficient numbers of follicles develop to allow for embryo transfer and subsequent pregnancy. PMID:22245528

  9. Expression and localization of aquaporin-1 on the apical membrane of enterocytes in the small intestine of bottlenose dolphins.

    PubMed

    Suzuki, Miwa

    2010-02-01

    The small and large intestines are primary sites for water intake in mammals. To reveal how water is absorbed in the intestines of cetaceans, histological and molecular-biological studies were performed on the small intestine of the bottlenose dolphin, Tursiops truncatus. In histological studies using fresh specimens, obvious villi and deep crypts of Lieberkühn, lined by abundant enterocytes with microvilli and goblet cells, were observed in the mucosa. Expressions and immunolocalizations of aquaporin-1 (AQP1), a member of the water-selective channel termed AQP, were also investigated in the intestine. By reverse transcriptional polymerase chain reaction and rapid amplification of cDNA ends using RNA extracted from the dolphins' small intestines, the full length of mRNA for AQP1 was sequenced. The deductive amino acid sequence for an open reading frame showed high homologies with other mammals' AQP1, and water permeability of the protein was certified by cRNA injection to Xenopus oocytes. Immunohistochemistry showed AQP1 distribution on the apical membrane of the enterocytes, especially in the crypts. These data suggest that AQP1 is a channel protein responsible for water absorption in the small intestine of dolphins.

  10. Tocopherol functions in photosynthetic organisms.

    PubMed

    Maeda, Hiroshi; DellaPenna, Dean

    2007-06-01

    During the past decade, the genes required for tocopherol (vitamin E) synthesis in plants and cyanobacteria have been identified. A series of mutants in which specific pathway steps are disrupted have been generated, providing new insights into tocopherol functions in photosynthetic organisms. Tocopherols are essential for controlling non-enzymatic lipid peroxidation during seed dormancy and seedling germination. Their absence results in elevated levels of malondialdehyde and phytoprostanes, and in inappropriate activation of plant defense responses. Surprisingly, tocopherol deficiency in mature leaves has limited consequences under most abiotic stresses, including high intensity light stress. The cell wall development of phloem transfer cells under cold conditions is, however, severely impaired in mature leaves of tocopherol-deficient mutants, indicating that tocopherols are required for proper adaptation of phloem loading at low temperatures.

  11. Differential expression and cytoplasm/membrane distribution of endoglin (CD105) in human tumour cell lines: Implications in the modulation of cell proliferation.

    PubMed

    Postiglione, L; Di Domenico, G; Caraglia, M; Marra, M; Giuberti, G; Del Vecchio, L; Montagnani, S; Macri, M; Bruno, E M; Abbruzzese, A; Rossi, G

    2005-05-01

    Endoglin (CD105, an accessory component of the TGF-beta receptor complex) expression and distribution on different human tumour cells and its role in cellular proliferation were evaluated. We examined: 1) sixteen human carcinoma cell lines, 2) eight human sarcoma cell lines, 3) five miscellaneous tumour cell lines. HECV (endothelial cells) were employed as a positive control for endoglin expression. Normal Human Dermal Fibroblasts (NHDF) and 293 cells (epithelial kidney cells) were used as normal controls for connective and epithelial tissues, respectively. The results showed that CD105 was poorly expressed in the majority of human carcinoma cells (10/16), whereas it was highly expressed in most human sarcoma cells (7/8), and differently expressed by miscellaneous tumour cell lines. These data reflect endoglin expression by the normal counterparts of tumour cell lines, i.e. NHDF and 293 cells. However, CD105 levels in sarcoma cell lines, even though consistently lower than in NHDF, were significantly higher than those observed in carcinoma cells. Interestingly, CD105 presented a strong expression in the cytoplasm of MDA-MB-453 (breast carcinoma), NPA (papillary thyroid carcinoma), COLO-853 (melanoma) and SaOS-2 (osteosarcoma), but was weakly expressed on their cell membrane. This differential expression in the cytoplasm and on the membrane of some tumour cells, suggests a complex mechanism of translocation for this protein. The analysis of clonal growth in soft agar of some cell lines, characterized by high CD105 expression, showed an increased colony formation potential that was antagonized by the addition of anti-CD105 blocking mAb. The results indicated that endoglin is differentially expressed in human carcinoma and sarcoma cells and its overexpression modulates the proliferative rate of human solid tumour cells. Moreover, these data suggest that CD105 is involved in the regulation of TGF-beta effects in human solid malignancies, and therefore it could play an

  12. 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

  13. Photosynthetic electron transport in thylakoids from cotton cultivars (Gossypium sp.) differing in tolerance to prometryn.

    PubMed

    Khan, R A; Molin, W T

    1996-09-01

    A method to determine photosynthetic electron transport in thylakoid membranes is described for Gossypium barbadense (cv. Pima S-7) and G. hirsutum (cv. DP 5415). These cultivars differed markedly in tolerance to prometryn, a PS II inhibitor. The rates of photosynthetic electron transport obtained were 245 μmole oxygen mg(-1) chl h(1). Plant age and leaf size influenced the activity of the thylakoid preparations. Thylakoids from leaves of plants 24 to 37 d and 50-70 mm in diameter had the highest activities; thylakoids from cotyledons, fully expanded leaves and young leaves had low activity. Thylakoids from both species had similar photosynthetic activities and I50's for prometryn, atrazine and diuron. Thus, tolerance to prometryn was not due to differential binding at D1 protein.

  14. Contributions of Respiratory and Photosynthetic Pathways during Growth of a Facultative Photoautotrophic Cyanobacterium, Aphanocapsa 6714 1

    PubMed Central

    Der-Vartanian, Maurice; Joset-Espardellier, Francoise; Astier, Chantal

    1981-01-01

    Comparison of the growth parameters and photosynthetic capacities of cells of Aphanocapsa 6714 under various growth conditions led to the following conclusions: (a), no enzymic regulation of CO2/glucose assimilation takes place in this strain; (b), functioning of photodependent phosphorylating pathways turns off oxidative ATP synthesis; (c), no efficient regulation of pigment synthesis exists in these cells; (d), most modulations of photosynthetic activities probably occur through structural modifications of the photosynthetic membranes (a small proportion of the pigments might appear as a nonintegrated pool in the cell and be sensitive to synthesis regulation); and (e), photosystem II activity would be dependent on light intensity in a discontinuous way, the consequence of this property being the appearance of two successive exponential phases during phototrophic growth in adequate light conditions. PMID:16662036

  15. Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane.

    PubMed

    Durrani, Faiza Gul; Gul, Roquyya; Sadaf, Saima; Akhtar, Muhammad Waheed

    2015-08-01

    This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25 °C on induction with 20 μM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40 % acetonitrile with ~90 % purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059 Da. Relatively high soluble protein yield of 65.3 mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield.

  16. 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.

  17. Protein expression of Fatty acid transporter 2 is polarized to the trophoblast basal plasma membrane and increased in placentas from overweight/obese women

    PubMed Central

    Lager, Susanne; Ramirez, Vanessa I.; Gaccioli, Francesca; Jang, Brian; Jansson, Thomas; Powell, Theresa L.

    2016-01-01

    Background Obese and overweight women are more likely to deliver a large infant or an infant with increased adiposity, however the underlying mechanisms are not well established. We tested the hypothesis that placental capacity to transport fatty acid is increased in overweight/obese women. Methods Fifty-seven pregnant women with body mass index (BMI) ranging from 18.4 to 54.3 kg/m2 and with uncomplicated term pregnancies were recruited for collection of blood samples and placental tissue. Maternal and fetal levels of non-esterified fatty acids (NEFAs) were measured in plasma. The expression and localization of CD36/fatty acid translocase (FAT), fatty acid transport protein (FATP)2, and FATP4 was determined in fixed placental tissue and in isolated syncytiotrophoblast plasma membranes from normal and high BMI mothers. Results Maternal and fetal plasma NEFA levels did not correlate (n=42). FATP2 and FATP4 expressions were approximately four-fold higher in the basal plasma membrane (BPM) compared to the microvillous membrane (P<0.001; n=7) per unit membrane protein. BPM expression of FATP2 correlated with maternal BMI (P<0.01; n=30); there was no association between CD36/FAT or FATP4 expression and maternal BMI. Conclusion The polarization of FATPs to the BPM will facilitate fatty acid transfer across the placenta. In overweight/obese pregnancies, the increased FATP2 expression could contribute to increased fatty acid delivery to the fetus and while we have no direct data we speculate that this could lead accelerated fetal growth or increased fat deposition. PMID:27016784

  18. Expression of membrane type 1 matrix metalloproteinase in papillomavirus-positive cells: role of the human papillomavirus (HPV) 16 and HPV8 E7 gene products.

    PubMed

    Smola-Hess, Sigrun; Pahne, Jenny; Mauch, Cornelia; Zigrino, Paola; Smola, Hans; Pfister, Herbert J

    2005-05-01

    Matrix metalloproteinases (MMPs) degrade extracellular matrix. They are involved in cellular proliferation, migration, angiogenesis, invasion and metastasis. MT-1 MMP, a membrane-bound MMP, is expressed in carcinomas of the uterine cervix in vivo. This type of cancer is associated with human papillomavirus (HPV) infection. Here it was shown that keratinocytes transformed with HPV16 or HPV18 in vitro, and HPV-positive cervical carcinoma cell lines, constitutively expressed MT-1 MMP. Expression of the E7 protein from the mucosal and cutaneous high-risk types HPV16 and HPV8, but not from the cutaneous low-risk type HPV1, was sufficient to induce MT-1 MMP expression in primary human keratinocytes and HaCaT cells. As a consequence, MMP-2 was activated. MT-1 MMP expression might play a role in the HPV life cycle by promoting proliferation of host cells and might contribute to their invasive phenotype during malignant progression.

  19. 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

  20. Epstein-Barr virus latent membrane protein-1 activates CD25 expression in lymphoma cells involving the NFkappaB pathway.

    PubMed

    Vockerodt, M; Tesch, H; Kube, D

    2001-12-01

    Epstein-Barr virus (EBV) is associated with several human malignancies including Burkitt's lymphoma (BL), Hodgkin's disease (HD) and nasopharyngeal carcinoma. A variety of cytokines and receptors have been described to be activated by EBV. Here we show that the IL-2 receptor (IL-2R) alpha-chain, which is weakly expressed on normal resting lymphoid cells, is activated by EBV. Comparison of EBV-negative BL cell lines and their EBV convertants showed an enhanced CD25 expression in EBV-positive BL cells. Transient expression of the oncogenic virus protein latent membrane protein-1 (LMP1) in L428 Hodgkin's lymphoma cells and in Burkitt's lymphoma cells (BL2, BL41, BL30) cells leads to enhanced CD25 expression. Both C-terminal activating regions (CTARs) of LMP1 are involved in CD25 activation. Inhibition of LMP1-mediated NFkappaB enhancement by a constitutive repressive form of IkappaB-alpha resulted in decreased CD25 surface expression, indicating that NFkappaB is involved in CD25 gene regulation. Furthermore, LMP1-mediated CD25 activation was associated with enhanced levels of the soluble form of CD25 (sCD25) in L428 Hodgkin's lymphoma cells but not in BL cells. LMP1 associated enhanced expression of membrane CD25 and soluble CD25 may have immunomodulatory functions and could be involved in biology of EBV-associated diseases.

  1. 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

  2. CD133 expression correlates with membrane beta-catenin and E-cadherin loss from human hair follicle placodes during morphogenesis.

    PubMed

    Gay, Denise L; Yang, Chao-Chun; Plikus, Maksim V; Ito, Mayumi; Rivera, Charlotte; Treffeisen, Elsa; Doherty, Laura; Spata, Michelle; Millar, Sarah E; Cotsarelis, George

    2015-01-01

    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 (AJs) is clearly required for budding. Snail-mediated downregulation of AJ component E-cadherin is important for placode budding in mice. Beta-catenin, another AJ component, has been more difficult to study owing 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 AJ dissolution.

  3. [Effects of light quality on photosynthetic pigment contents and photosynthetic characteristics of peanut seedling leaves].

    PubMed

    Yan, Meng-Meng; Wang, Ming-Lun; Wang, Hong-Bo; Wang, Yue-Fu; Zhao, Chang-Xing

    2014-02-01

    This study explored the effects of different light quality on photosynthetic pigment contents and photosynthetic characteristics of peanut (Qinhua 6) seedling leaves. The results showed that, compared with natural light, blue light (445-470 nm) could significantly improve the specific leaf area (SLA), chlorophyll a/b value and carotenoid content of peanut seedlings. Meanwhile, the net photosynthetic rate, stomatal conductance, and transpiration rate were higher, the intercellular CO2 content was lower, and the photosynthetic efficiency was improved significantly under blue light. Red light (610-660 nm) could improve the chlorophyll content significantly, and reduce SLA, chlorophyll a/b value and carotenoid content, with a lower photosynthetic efficiency than natural light. Green light (515-520 nm) and yellow light (590-595 nm) were not conducive to photosynthetic pigment accumulation of leaves, and significantly inhibited leaf photosynthesis of peanut seedlings.

  4. Photosynthetic limitations and volatile and non-volatile isoprenoids in the poikilochlorophyllous resurrection plant Xerophyta humilis during dehydration and rehydration.

    PubMed

    Beckett, Megan; Loreto, Francesco; Velikova, Violeta; Brunetti, Cecilia; Di Ferdinando, Martina; Tattini, Massimiliano; Calfapietra, Carlo; Farrant, Jill M

    2012-12-01

    We investigated the photosynthetic limitations occurring during dehydration and rehydration of Xerophyta humilis, a poikilochlorophyllous resurrection plant, and whether volatile and non-volatile isoprenoids might be involved in desiccation tolerance. Photosynthesis declined rapidly after dehydration below 85% relative water content (RWC). Raising intercellular CO(2) concentrations during desiccation suggest that the main photosynthetic limitation was photochemical, affecting energy-dependent RuBP regeneration. Imaging fluorescence confirmed that both the number of photosystem II (PSII) functional reaction centres and their efficiency were impaired under progressive dehydration, and revealed the occurrence of heterogeneous photosynthesis during desiccation, being the basal leaf area more resistant to the stress. Full recovery in photosynthetic parameters occurred on rehydration, confirming that photosynthetic limitations were fully reversible and that no permanent damage occurred. During desiccation, zeaxanthin and lutein increased only when photosynthesis had ceased, implying that these isoprenoids do not directly scavenge reactive oxygen species, but rather protect photosynthetic membranes from damage and consequent denaturation. X. humilis was found to emit isoprene, a volatile isoprenoid that acts as a membrane strengthener in plants. Isoprene emission was stimulated by drought and peaked at 80% RWC. We surmise that isoprene and non-volatile isoprenoids cooperate in reducing membrane damage in X. humilis, isoprene being effective when desiccation is moderate while non-volatile isoprenoids operate when water deficit is more extreme.

  5. 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

  6. Iron regulated outer membrane proteins of Escherichia coli: variations in expression due to the chelator used to restrict the availability of iron.

    PubMed

    Chart, H; Buck, M; Stevenson, P; Griffiths, E

    1986-05-01

    Iron restriction was induced in Escherichia coli O 111, E. coli O 164 and E. coli C by growing the organisms in trypticase soy broth containing ovotransferrin, desferal, EDDA (ethylenediamine-dihydroxyphenylacetic acid) or alpha,alpha'-dipyridyl. There were marked qualitative and quantitative differences in the iron regulated outer membrane proteins expressed in the presence of the various iron chelators. Differences in the kinetics of growth were also noted. E. coli C was devoid of a ferric enterobactin iron uptake system.

  7. Mutagenesis of a novel gene in the prcA-prtP protease locus affects expression of Treponema denticola membrane complexes.

    PubMed

    Bian, Xue-Lin; Wang, Hong-Tao; Ning, Yu; Lee, Si Young; Fenno, J Christopher

    2005-02-01

    A novel gene was identified in the Treponema denticola prcA-prtP protease operon. Strains with mutations in either the prcA-prtP or the msp region showed altered expression of a product(s) of the other locus. Together, these results provide information on the assembly of outer membrane complexes involved in T. denticola interaction with host cells and tissue.

  8. Mutagenesis of a Novel Gene in the prcA-prtP Protease Locus Affects Expression of Treponema denticola Membrane Complexes

    PubMed Central

    Bian, Xue-lin; Wang, Hong-tao; Ning, Yu; Lee, Si Young; Fenno, J. Christopher

    2005-01-01

    A novel gene was identified in the Treponema denticola prcA-prtP protease operon. Strains with mutations in either the prcA-prtP or the msp region showed altered expression of a product(s) of the other locus. Together, these results provide information on the assembly of outer membrane complexes involved in T. denticola interaction with host cells and tissue. PMID:15664975

  9. Immunoreactivity and differential developmental expression of known and putative Chlamydia trachomatis membrane proteins for biologically variant serovars representing distinct disease groups.

    PubMed

    Gomes, João P; Hsia, Ru-ching; Mead, Sally; Borrego, Maria J; Dean, Deborah

    2005-03-01

    Chlamydia trachomatis is an intracellular bacterium that causes ocular and urogenital diseases worldwide. Membrane proteins have only been partially characterized, and the discovery of a nine-member polymorphic membrane protein gene family has enhanced interest in defining their function. We previously reported two putative insertion sequence-like elements in pmpC for biovariant Ba and one each for G and L2, suggesting horizontal gene transfer. Because of this and the tissue tropism differences for these biovariants, we analyzed by quantitative real-time RT-PCR pmpC expression relative to immunogenic protein genes ompA, groEL and gseA throughout development. Sera from infected adolescents were reacted by immunoblot against recombinant (r)PmpC and rMOMP. ompA and groEL revealed different developmental transcriptome profiles among the biovariants. pmpC expression occurred at 2 h, peaked at 18 for L2 (at 24 for Ba and G), with the highest mRNA levels throughout development for L2. pmpC expression as a function of time paralleled ompA expression with higher mRNA levels compared with groEL later in development. Only sera from D-, E- and G-infected patients reacted to rPmpC; all infected patients reacted to rMOMP. pmpC expression during logarithmic growth suggests a role in membrane building and/or integrity, which is supported by the presence of a signal peptidase and C-terminal phenylalanine in PmpC. Because phylogenetic analyses of pmpC segregate serovars according to tissue tropism, we speculate that biovariant transcriptome differences may contribute to this tropism. The heterogeneous biovariant pmpC expression throughout development and differential PmpC immunoreactivity also suggest a role for pmpC in antigenic variation.

  10. Hybrid system of semiconductor and photosynthetic protein

    NASA Astrophysics Data System (ADS)

    Kim, Younghye; Shin, Seon Ae; Lee, Jaehun; Yang, Ki Dong; Nam, Ki Tae

    2014-08-01

    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.

  11. 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.

  12. Design principles of photosynthetic light-harvesting.

    PubMed

    Fleming, Graham R; Schlau-Cohen, Gabriela S; Amarnath, Kapil; Zaks, Julia

    2012-01-01

    Photosynthetic organisms are capable of harvesting solar energy with near unity quantum efficiency. Even more impressively, this efficiency can be regulated in response to the demands of photosynthetic reactions and the fluctuating light-levels of natural environments. We discuss the distinctive design principles through which photosynthetic light-harvesting functions. These emergent properties of photosynthesis appear both within individual pigment-protein complexes and in how these complexes integrate to produce a functional, regulated apparatus that drives downstream photochemistry. One important property is how the strong interactions and resultant quantum coherence, produced by the dense packing of photosynthetic pigments, provide a tool to optimize for ultrafast, directed energy transfer. We also describe how excess energy is quenched to prevent photodamage under high-light conditions, which we investigate through theory and experiment. We conclude with comments on the potential of using these features to improve solar energy devices.

  13. 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

  14. Increased levels of soluble CD226 in sera accompanied by decreased membrane CD226 expression on peripheral blood mononuclear cells from cancer patients

    PubMed Central

    Xu, Zhuwei; Zhang, Tao; Zhuang, Ran; Zhang, Yun; Jia, Wei; Song, Chaojun; Yang, Kun; Yang, Angang; Jin, Boquan

    2009-01-01

    Background As a cellular membrane triggering receptor, CD226 is involved in the NK cell- or CTL-mediated lysis of tumor cells of different origin, including freshly isolated tumor cells and tumor cell lines. Here, we evaluated soluble CD226 (sCD226) levels in sera, and membrane CD226 (mCD226) expression on peripheral blood mononuclear cells (PBMC) from cancer patients as well as normal subjects, and demonstrated the possible function and origin of the altered sCD226, which may provide useful information for understanding the mechanisms of tumor escape and for immunodiagnosis and immunotherapy. Results Soluble CD226 levels in serum samples from cancer patients were significantly higher than those in healthy individuals (P < 0.001), while cancer patients exhibited lower PBMC mCD226 expression than healthy individuals (P < 0.001). CD226-Fc fusion protein could significantly inhibit the cytotoxicity of NK cells against K562 cells in a dose-dependent manner. Furthermore, three kinds of protease inhibitors could notably increase mCD226 expression on PMA-stimulated PBMCs and Jurkat cells with a decrease in the sCD226 level in the cell culture supernatant. Conclusion These findings suggest that sCD226 might be shed from cell membranes by certain proteases, and, further, sCD226 may be used as a predictor for monitoring cancer, and more important, a possible immunotherapy target, which may be useful in clinical application. PMID:19490613

  15. 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.

  16. Efficiency of light harvesting in a photosynthetic bacterium adapted to different levels of light.

    PubMed

    Timpmann, Kõu; Chenchiliyan, Manoop; Jalviste, Erko; Timney, John A; Hunter, C Neil; Freiberg, Arvi

    2014-10-01

    In this study, we use the photosynthetic purple bacterium Rhodobacter sphaeroides to find out how the acclimation of photosynthetic apparatus to growth conditions influences the rates of energy migration toward the reaction center traps and the efficiency of charge separation at the reaction centers. To answer these questions we measured the spectral and picosecond kinetic fluorescence responses as a function of excitation intensity in membranes prepared from cells grown under different illumination conditions. A kinetic model analysis yielded the microscopic rate constants that characterize the energy transfer and trapping inside the photosynthetic unit as well as the dependence of exciton trapping efficiency on the ratio of the peripheral LH2 and core LH1 antenna complexes, and on the wavelength of the excitation light. A high quantum efficiency of trapping over 80% was observed in most cases, which decreased toward shorter excitation wavelengths within the near infrared absorption band. At a fixed excitation wavelength the efficiency declines with the LH2/LH1 ratio. From the perspective of the ecological habitat of the bacteria the higher population of peripheral antenna facilitates growth under dim light even though the energy trapping is slower in low light adapted membranes. The similar values for the trapping efficiencies in all samples imply a robust photosynthetic apparatus that functions effectively at a variety of light intensities.

  17. Epstein Barr-Virus Latent Membrane Protein-2A-Induced ΔNp63α Expression is Associated with Impaired Epithelial Cell Differentiation

    PubMed Central

    Fotheringham, Julie A.; Mazzucca, Stephanie; Raab-Traub, Nancy

    2010-01-01

    Epstein-Barr virus (EBV) is an oncogenic γ-herpes virus associated with malignancies that develop in both lymphoid and epithelial cells including nasopharyngeal carcinoma (NPC). The EBV protein latent membrane protein 2A (LMP2A) is expressed in NPC and can modulate epithelial proliferation, transformation, and differentiation, and as such, may promote malignancy. A key regulator of epithelial cell differentiation is the transcription factor p63, a member of the p53 family. This study examines the potential contribution of p63 to LMP2A-mediated inhibition of epithelial differentiation. Stable expression of LMP2A increased the protein level and stability of the ΔNp63α isoform, and in two epithelial cell lines, LMP2A interacted with ΔNp63α under stable and transient expression systems. LMP2A and ΔNp63α were localized to the cytoplasm and nuclear membrane and co-immunoprecipitated in the same fractions. Following induction of epithelial cell differentiation by calcium, expression of differentiation markers was impaired in both ΔNp63α and LMP2 expressing cells. Induction of p63α, association of p63α with LMP2A, and impairment of differentiation required the PY and ITAM signaling motif of LMP2A. By associating with and being regulated by LMP2A,ΔNp63α may function as a unique regulator of LMP2A effects on epithelial differentiation and contribute to EBV-associated epithelial cancers. PMID:20498633

  18. Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

    PubMed Central

    2012-01-01

    The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. PMID:22292497

  19. Calorie restriction reduces pinealectomy-induced insulin resistance by improving GLUT4 gene expression and its translocation to the plasma membrane.

    PubMed

    Zanquetta, Melissa M; Seraphim, Patricia M; Sumida, Doris H; Cipolla-Neto, Jose; Machado, Ubiratan F

    2003-10-01

    The present study aimed to investigate insulin sensitivity and GLUT4 expression protein in pinealectomized rats, as well as to determining the effects of melatonin and calorie restriction on the changes induced by pinealectomy. Wistar rats were pinealectomized (Pinx) or sham operated (Sham), and studied 30 days later. Melatonin replacement treatment (50 g/100 g body weight) was continued for 30 days after pinealectomy. Calorie restriction was performed by offering 60% of the standard food intake. In vivo insulin sensitivity was evaluated using the glucose disappearance constant (kITT) during an insulin tolerance test, and GLUT4 mRNA and protein were assessed by Northern and Western blotting, respectively. The in vitro effect of melatonin on GLUT4 protein content in plasma membrane was investigated in adipocytes isolated from intact rats. Compared with Sham rats, Pinx rats showed decreased kITT (40%), GLUT4 expression in white adipose tissue (WAT, approximately 70%), and unchanged GLUT4 expression in skeletal muscle. Melatonin treatment in Pinx rats restored the kITT and GLUT4 protein to control values. No in vitro effects of melatonin (10-9 m) upon GLUT4 protein were observed. Calorie restriction of Pinx rats increased their kITT value ( approximately 40%), total GLUT4 protein content ( approximately 240%) and its translocation to the plasma membrane ( approximately 80%) in WAT. The results show that pinealectomy, for lack of melatonin, decreased insulin sensitivity as well as GLUT4 gene expression. Calorie restriction improved insulin sensitivity in Pinx rats, and this was related to increased GLUT4 gene expression and insulin-induced GLUT4 translocation to the plasma membrane in WAT.

  20. 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

  1. 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.

  2. 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

    2015-05-06

    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.

  3. Involvement of plasma membrane redox activity and calcium homeostasis in the UV-B and UV-A/blue light induction of gene expression in Arabidopsis.

    PubMed Central

    Long, J C; Jenkins, G I

    1998-01-01

    UV and blue light are important regulators of plant gene expression and development. We investigated the signal transduction processes involved in the induction of chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) gene expression by UV-B and UV-A/blue light in an Arabidopsis cell suspension culture. Experiments with electron transport inhibitors indicated that plasma membrane redox activity is involved in both signal transduction pathways. Calcium ionophore treatment stimulated expression of the TOUCH3 gene, and this induction was strongly antagonized by UV-A/blue and UV-B light, suggesting that both light qualities may promote calcium efflux from the cytosol. Consistent with this hypothesis, experiments with specific inhibitors indicated that UV-B and UV-A/blue light regulate calcium levels in a cytosolic pool in part via the action of specific Ca2+-ATPases. On the basis of these and previous findings, we propose that plasma membrane redox activity, initiated by photoreception, is coupled to the regulation of calcium release from an intracellular store, generating a calcium signal that is required to induce CHS expression. PMID:9836746

  4. Evidence that the Essential Response Regulator YycF in Streptococcus pneumoniae Modulates Expression of Fatty Acid Biosynthesis Genes and Alters Membrane Composition†

    PubMed Central

    Mohedano, M. Luz; Overweg, Karin; de la Fuente, Alicia; Reuter, Mark; Altabe, Silvia; Mulholland, Francis; de Mendoza, Diego; López, Paloma; Wells, Jerry M.

    2005-01-01

    The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism. PMID:15774879

  5. 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.

  6. Lipid-protein interactions in Escherichia coli membranes over-expressing the sugar-H(+) symporter, GalP EPR of spin-labelled lipids.

    PubMed

    Hubert, Anne; Henderson, Peter J F; Marsh, Derek

    2003-04-01

    The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, lipid-protein interactions were studied in gradient-purified inner membranes by using spin-label electron paramagnetic resonance (EPR) spectroscopy. Phosphatidylethanolamine, -glycerol, -choline and -serine, in addition to phosphatidic and stearic acids, were spin-labelled at the 14 C-atom of the sn-2 chain. EPR spectra of these spin labels at probe amounts in GalP membranes consist of two components. One component corresponds to a lipid population whose motion is restricted by direct interaction with the transmembrane sections of the integral protein. The other component corresponds to a lipid population with greater chain mobility, and is similar to the single-component EPR spectrum of the spin-labelled lipids in membranes of E. coli lipid extract. Quantitation of the protein-interacting spin-label component allows determination of the stoichiometry and selectivity of lipid-protein interactions. On average, approximately 20 mol of lipid are motionally restricted per 52 kDa of protein in GalP membranes. At the pH of the transport assay, there is relatively little selectivity between the different phospholipids tested. Only stearic acid displays a stronger preferential interaction with this protein.

  7. 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.

  8. Expression of matrix metalloproteinases during rat skin wound healing: evidence that membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase A.

    PubMed

    Okada, A; Tomasetto, C; Lutz, Y; Bellocq, J P; Rio, M C; Basset, P

    1997-04-07

    Skin wound healing depends on cell migration and extracellular matrix remodeling. Both processes, which are necessary for reepithelization and restoration of the underlying connective tissue, are believed to involve the action of extracellular proteinases. We screened cDNA libraries and we found that six matrix metalloproteinase genes were highly expressed during rat skin wound healing. They were namely those of stromelysin 1, stromelysin 3, collagenase 3, gelatinase A (GelA), gelatinase B, and membrane type-1 matrix metalloproteinase (MT1-MMP). The expression kinetics of these MMP genes, the tissue distribution of their transcripts, the results of cotransfection experiments in COS-1 cells, and zymographic analyses performed using microdissected rat wound tissues support the possibility that during cutaneous wound healing pro-GelA and pro-gelatinase B are activated by MT1-MMP and stromelysin 1, respectively. Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. Nature (Lond.). 370: 61-65), our finding that GelA and MT1-MMP transcripts were expressed in stromal cells exhibiting a similar tissue distribution suggests that MT1-MMP activates pro-GelA at the stromal cell surface. This possibility is further supported by our observation that the processing of pro-GelA to its mature form correlated to the detection of MT1-MMP in cell membranes of rat fibroblasts expressing the MT1-MMP and GelA genes. These observations, together with the detection of high levels of the mature GelA form in the granulation tissue but not in the regenerating epidermis, suggest that MT1-MMP and GelA contribute to the restoration of connective tissue during rat skin wound healing.

  9. 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.

  10. 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

  11. The 4p16.3 Parkinson Disease Risk Locus Is Associated with GAK Expression and Genes Involved with the Synaptic Vesicle Membrane.

    PubMed

    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.

  12. Increased Expression of CCN2, Epithelial Membrane Antigen, and Fibroblast Activation Protein in Hepatocellular Carcinoma with Fibrous Stroma Showing Aggressive Behavior

    PubMed Central

    Yoo, Jeong Eun; Ko, Jung Eun; Lee, Jee San; Kim, Hyunki; Choi, Jin Sub; Park, Young Nyun

    2014-01-01

    Tumor behavior is affected by the tumor microenvironment, composed of cancer-associated fibroblasts (CAFs). Meanwhile, hepatocellular carcinomas (HCC) with fibrous stroma reportedly exhibit aggressive behavior suggestive of tumor-stroma interaction. However, evidence of the crosstalk remains unclear. In this study, CCN2, epithelial membrane antigen (EMA), fibroblast activation protein (FAP), and keratin 19 (K19) expression was studied in 314 HCCs (cohort 1), 42 scirrhous HCCs (cohort 2), and 36 chronic hepatitis/cirrhosis specimens by immunohistochemistry. Clinicopathological parameters were analyzed according to the expressions of these markers. In tumor epithelial cells from cohort 1, CCN2 and EMA were expressed in 15.3% and 17.2%, respectively, and their expressions were more frequent in HCCs with fibrous stroma (≥5% of tumor area) than those without (P<0.05 for all); CCN2 expression was well correlated with K19 and EMA expression. In tumor stromal cells, FAP expression was found in 6.7%. In cohort 2, CCN2, EMA, and FAP expression was noted in 40.5%, 40.5%, and 66.7%, respectively, which was more frequent than that in cohort 1 (P<0.05 for all). Additionally, EMA expression was associated with the expression of K19, CCN2, and FAP (P<0.05 for all); EMA expressing tumor epithelial cells showed a topographic closeness to FAP-expressing CAFs. Analysis of disease-free survival revealed CCN2 expression to be a worse prognostic factor in both cohort 1 (P = 0.005) and cohort 2 (P = 0.023), as well as EMA as a worse prognostic factor in cohort 2 (P = 0.048). In conclusion, expression of CCN2, EMA, and FAP may be involved in the activation of CAFs in HCC, giving rise to aggressive behavior. Significant correlation between EMA-expressing tumor cells and FAP-expressing CAFs and their topographic closeness suggests possible cross-talk between tumor epithelial cells and stromal cells in the tumor microenvironment of HCC. PMID:25126747

  13. 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

  14. Progestin treatment does not affect expression of cytokines, steroid receptors, oxytocin receptor, and cyclooxygenase 2 in fetal membranes and endometrium from pony mares at parturition.

    PubMed

    Palm, F; Walter, I; Nowotny, N; Budik, S; Helmreich, M; Aurich, C

    2013-01-01

    In most mammalian species, progestins have a major function in maintaining pregnancy. In humans, the physiologic initiation of parturition bears similarities with inflammatory processes and anti-inflammatory effects of progestins have been suggested to postpone birth until term. To examine if comparable effects exist in the horse, mares were treated with the synthetic progestin altrenogest from day 280 of gestation until parturition (N = 5) or were left untreated as controls (N = 7). Tissue from the amnion (AMN), allantochorion (AC), and endometrium (EM) was collected at foaling and mRNA expression of interleukin (IL)-6 and -8, cyclooxygenase 2 (COX2), estrogen receptor (ER) α, progesterone receptor, and oxytocin receptor (OTR) was analyzed. Leukocytes, steroid receptors, COX2, and OTR were also investigated by histology and immunohistochemistry. Expression of mRNA for IL-6 was higher in AMN and EM versus AC (P < 0.01). Expression of IL-8 was higher in AMN than AC and EM (P < 0.001). Steroid receptors and OTR were highly expressed in EM but not in AMN and AC (P < 0.001). Expression of COX2 was most pronounced in AC whereas IL expression was not upregulated in AC. No differences in mRNA expression existed between altrenogest-treated and control animals. Endometrial polymorphonuclear leukocytes were increased in altrenogest-treated mares. Epithelial cells of all tissues, except AC chorionic villi stained progesterone receptor-positive. Staining for ER was more pronounced in the amnion facing epithelium of the AC in altrenogest-treated versus control animals (P < 0.01). In conclusion, COX2 is highly expressed in the AC. The fetal membranes thus might play a role in the onset of labor in the horse. Altrenogest did not affect gene expression in the AMN, AC, and EM but had localized effects on inflammatory cells and ER expression. No anti-inflammatory effects of altrenogest in healthy, late pregnant pony mares could be detected.

  15. The occurrence of the psbS gene product in Chlamydomonas reinhardtii and in other photosynthetic organisms and its correlation with energy quenching.

    PubMed

    Bonente, Giulia; Passarini, Francesca; Cazzaniga, Stefano; Mancone, Carmine; Buia, Maria Cristina; Tripodi, Marco; Bassi, Roberto; Caffarri, Stefano

    2008-01-01

    To avoid photodamage, photosynthetic organisms have developed mechanisms to evade or dissipate excess energy. Lumen overacidification caused by light-induced electron transport triggers quenching of excited chlorophylls and dissipation of excess energy into heat. In higher plants participation of the PsbS protein as the sensor of low lumenal pH was clearly demonstrated. Although light-dependent energy quenching is a property of all photosynthetic organisms, large differences in amplitude and kinetics can be observed thus raising the question whether a single common mechanism is in action. We performed a detailed study of PsbS expression/accumulation in Chlamydomonas reinhardtii and investigated its accumulation in other algae and plants. We showed that PsbS cannot be detected in Chlamydomonas under a wide range of growth conditions. Overexpression of the endogenous psbs gene showed that the corresponding protein could not be addressed to the thylakoid membranes. Survey of different unicellular green algae showed no accumulation of anti-PsbS reactive proteins differently from multicellular species. Nevertheless, some unicellular species exhibit high energy quenching activity, suggesting that a PsbS-independent mechanism is activated. By correlating growth habitat and PsbS accumulation in different species, we suggest that during the evolution the light environment has been a determinant factor for the conservation/loss of the PsbS function.

  16. Expression and purification of the recombinant membrane protein YidC: a case study for increased stability and solubility.

    PubMed

    Martinez Molina, Daniel; Lundbäck, Anna-Karin; Niegowski, Damian; Eshaghi, Said

    2008-11-01

    YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, translocation and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 microg of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high concentrations and that was stable for weeks at +4 degrees C. The isolated protein is thus well suited for structural studies.

  17. Effects of selenium on peripheral blood mononuclear cell membrane fluidity, interleukin-2 production and interleukin-2 receptor expression in patients with chronic hepatitis

    PubMed Central

    He, Shui-Xiang; Wu, Bing; Chang, Xin-Ming; Li, Hong-Xia; Qiao, Wen

    2004-01-01

    AIM: To study the effect of selenium on peripheral blood mononuclear cell (PBMC) membrane fluidity and immune function in patients with chronic hepatitis. METHODS: PBMCs were pretreated with selenium (1.156 × 10-7 mol/L) for 6 h in vitro or extracted directly from patients after administration of selenium-yeast continuously for 8-12 wk (200 μg/d), and then exposed to Con-A for 48 h. The membrane fluidity, interleukin-2 (IL-2) production and interleukin-2 receptor (IL-2R) expression in PBMCs and malondialdehyde (MDA) concentration in medium and lipid peroxide (LPO) in plasma were determined. RESULTS: The PBMC membrane fluidity, IL-2 production and IL-2R expression in patients with chronic hepatitis were significantly lower than those in healthy blood donators (particle adhesive degree R, 0.17 ± 0.01 vs 0.14 ± 0.01, P < 0.01; IL-2, 40.26 ± 9.55 vs 72.96 ± 11.36, P < 0.01; IL-2R, 31.05 ± 5.09 vs 60.58 ± 10.56, P < 0.01), and the MDA concentration in medium in patients with chronic hepatitis was significantly higher than that in healthy blood donators (1.44 ± 0.08 vs 0.93 ± 0.08, P < 0.01). Both in vitro and in vivo administration of selenium could reverse the above parameters. CONCLUSION: Supplement of selenium can suppress lipid peroxidation, and improve PBMC membrane fluidity and immune function in patients with chronic hepatitis. PMID:15526380

  18. 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.

  19. 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).

  20. The transforming domain alone of the latent membrane protein of Epstein-Barr virus is toxic to cells when expressed at high levels.

    PubMed Central

    Hammerschmidt, W; Sugden, B; Baichwal, V R

    1989-01-01

    A previously unrecognized activity has been associated with the product of the BNLF-1 gene of Epstein-Barr virus. This gene encodes the latent membrane protein of Epstein-Barr virus. When the gene was expressed at high levels, it was toxic to all cell lines tested, which included six human B-lymphoid lines as well as BALB/3T3, 143/EBNA-1, and HEp-2 cells. The BNLF-1 gene was previously shown to induce anchorage-independent and tumorigenic growth in Rat-1 and BALB/3T3 cells. We demonstrate here that only those mutations in the BNLF-1 gene that score positively in the anchorage-independent growth assay were cytotoxic when expressed at high levels. It is therefore possible that the same activities of the latent membrane protein that are necessary to induce anchorage-independent growth of some rodent cell lines also confer toxicity to many cell lines when expressed at high levels. Images PMID:2542565

  1. 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

  2. A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp).

    PubMed

    Stross, Claudia; Kluge, Stefanie; Weissenberger, Katrin; Winands, Elisabeth; Häussinger, Dieter; Kubitz, Ralf

    2013-11-15

    The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ~30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.

  3. 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...

  4. Senescence Marker Protein 30 (SMP30) Expression in Eukaryotic Cells: Existence Of Multiple Species and Membrane Localization

    DTIC Science & Technology

    2011-02-01

    1000 dilution) or mouse-a-actin monoclonal antibody conjugated with Horse Radish Peroxidase (1:50,000 dilution). The membrane was then washed with...was Goat-anti-rabbit conjugated with Horse Radish Peroxidase (KPL, Inc, Rockville, MD; 1:2500 dilution). No secondary antibody was used in the case of

  5. A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

    PubMed

    Minkoff, Benjamin B; Makino, Shin-Ichi; Haruta, Miyoshi; Beebe, Emily T; Wrobel, Russell L; Fox, Brian G; Sussman, Michael R

    2017-04-07

    There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.

  6. 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.

  7. 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.

  8. A remotely sensed pigment index reveals photosynthetic phenology in evergreen conifers

    PubMed Central

    Huemmrich, K. Fred; Ensminger, Ingo; Garrity, Steven; Noormets, Asko; Peñuelas, Josep

    2016-01-01

    In evergreen conifers, where the foliage amount changes little with season, accurate detection of the underlying “photosynthetic phenology” from satellite remote sensing has been difficult, presenting challenges for global models of ecosystem carbon uptake. Here, we report a close correspondence between seasonally changing foliar pigment levels, expressed as chlorophyll/carotenoid ratios, an