Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing.

Science.gov (United States)

Izawa, Shingo; Ikeda, Kayo; Miki, Takeo; Wakai, Yoshinori; Inoue, Yoshiharu

2010-09-01

Although ethanol and osmotic stress affect the vacuolar morphology of Saccharomyces cerevisiae, little information is available about changes in vacuolar morphology during the processes of wine making and Japanese sake (rice wine) brewing. Here, we elucidated changes in the morphology of yeast vacuoles using Zrc1p-GFP, a vacuolar membrane protein, so as to better understand yeast physiology during the brewing process. Wine yeast cells (OC-2 and EC1118) contained highly fragmented vacuoles in the sake mash (moromi) as well as in the grape must. Although sake yeast cells (Kyokai no. 9 and no. 10) also contained highly fragmented vacuoles during the wine-making process, they showed quite a distinct vacuolar morphology during sake brewing. Since the environment surrounding sake yeast cells in the sake mash did not differ much from that surrounding wine yeast cells, the difference in vacuolar morphology during sake brewing between wine yeast and sake yeast was likely caused by innate characters.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

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Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

2015-12-01

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. © 2015 American Society of Plant Biologists. All rights reserved.

TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

NARCIS (Netherlands)

Appelhagen, I.; Nordholt, N.; Seidel, T.; Spelt, K.; Koes, R.; Quattrochio, F.; Sagasser, M.; Weisshaar, B.

2015-01-01

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+)

Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants

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Pehlivan, Necla; Sun, Li; Jarrett, Philip; Yang, Xiaojie; Mishra, Neelam; Chen, Lin; Kadioglu, Asim; Shen, Guoxin; Zhang, Hong

2016-01-01

The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na+/H+) antiporter that transports Na+ into the vacuole and exports H+ into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na+/H+ antiporter that exports Na+ to the extracellular space and imports H+ into the plant cell. Plants rely on these enzymes either to keep Na+ out of the cell or to sequester Na+ into vacuoles to avoid the toxic level of Na+ in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops. PMID:26985021

Vacuolar structures can be identified by AFM elasticity mapping

International Nuclear Information System (INIS)

Riethmueller, Christoph; Schaeffer, Tilman E.; Kienberger, Ferry; Stracke, Werner; Oberleithner, Hans

2007-01-01

Fluid-filled organelles like vesicles, endosomes and pinosomes are inevitable parts of cellular signalling and transport. Endothelial cells, building a barrier between blood and tissue, can form vacuolar organelles. These structures are implicated in upregulated fluid transport across the endothelium under inflammatory conditions. Vacuolar organelles have been described by transmission electron microscopy so far. Here, we present a method that images and mechanically characterizes intracellular structures in whole cells by atomic force microscopy (AFM). After crosslinking the cellular proteins with the fixative glutaraldehyde, plasma membrane depressions become observable, which are scattered around the cell nucleus. Nanomechanical analysis identifies them as spots of reduced stiffness. Scanning electron microscopy confirms their pit-like appearance. In addition, fluorescence microscopy detects an analogous pattern of protein-poor spots, thereby confirming mechanical rigidity to arise from crosslinked proteins. This AFM application opens up a mechanical dimension for the investigation of intracellular organelles

Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants.

Science.gov (United States)

Pehlivan, Necla; Sun, Li; Jarrett, Philip; Yang, Xiaojie; Mishra, Neelam; Chen, Lin; Kadioglu, Asim; Shen, Guoxin; Zhang, Hong

2016-05-01

The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na(+)/H(+)) antiporter that transports Na(+) into the vacuole and exports H(+) into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na(+)/H(+) antiporter that exports Na(+) to the extracellular space and imports H(+) into the plant cell. Plants rely on these enzymes either to keep Na(+) out of the cell or to sequester Na(+) into vacuoles to avoid the toxic level of Na(+) in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

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Carvalho TMU

1999-01-01

Full Text Available Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV. In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37ºC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

Vacuolar Chloride Fluxes Impact Ion Content and Distribution during Early Salinity Stress1

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Baetz, Ulrike; Tohge, Takayuki; Martinoia, Enrico; De Angeli, Alexis

2016-01-01

The ability to control the cytoplasmic environment is a prerequisite for plants to cope with changing environmental conditions. During salt stress, for instance, Na+ and Cl− are sequestered into the vacuole to help maintain cytosolic ion homeostasis and avoid cellular damage. It has been observed that vacuolar ion uptake is tied to fluxes across the plasma membrane. The coordination of both transport processes and relative contribution to plant adaptation, however, is still poorly understood. To investigate the link between vacuolar anion uptake and whole-plant ion distribution during salinity, we used mutants of the only vacuolar Cl− channel described to date: the Arabidopsis (Arabidopsis thaliana) ALMT9. After 24-h NaCl treatment, almt9 knock-out mutants had reduced shoot accumulation of both Cl− and Na+. In contrast, almt9 plants complemented with a mutant variant of ALMT9 that exhibits enhanced channel activity showed higher Cl− and Na+ accumulation. The altered shoot ion contents were not based on differences in transpiration, pointing to a vacuolar function in regulating xylem loading during salinity. In line with this finding, GUS staining demonstrated that ALMT9 is highly expressed in the vasculature of shoots and roots. RNA-seq analysis of almt9 mutants under salinity revealed specific expression profiles of transporters involved in long-distance ion translocation. Taken together, our study uncovers that the capacity of vacuolar Cl− loading in vascular cells plays a crucial role in controlling whole-plant ion movement rapidly after onset of salinity. PMID:27503602

Characterization of Gene Candidates for Vacuolar Sodium Transport from Hordeum Vulgare

KAUST Repository

Scheu, Arne Hagen August

2017-05-01

Soil salinity is a major abiotic stress for land plants, and multiple mechanisms of salt tolerance have evolved. Tissue tolerance is one of these mechanisms, which involves the sequestration of sodium into the vacuole to retain low cytosolic sodium concentrations. This enables the plant to maintain cellular functions, and ultimately maintain growth and yield. However, the molecular components involved in tissue tolerance remain elusive. Several candidate genes for vacuolar sodium sequestration have recently been identified by proteome analysis of vacuolar membranes purified from the salt-tolerant cereal Hordeum vulgare (barley). In this study, I aimed to characterize these candidates in more detail. I successfully cloned coding sequences for the majority of candidate genes with primers designed based on the barley reference genome sequence. During the course of this study a newer genome sequence with improved annotations was published, to which I also compared my observations. To study the candidate genes, I used the heterologous expression system Saccharomyces cerevisiae (yeast). I used several salt sensitive yeast strains (deficient in intrinsic sodium transporters) to test whether the candidate genes would affect their salt tolerance by mediating the sequestration of sodium into the yeast vacuole. I observed a reduction in growth upon expression for several of the gene candidate under salt-stress conditions. However, confocal microscopy suggests that most gene products are subject to degradation, and did not localize to the vacuolar membrane (tonoplast). Therefore, growth effects cannot be linked to protein function without further evidence. Various potential causes are discussed, including inaccuracies in the genome resource used as reference for primer design and issues inherent to the model system. Finally, I make suggestions on how to proceed to further characterize the candidate genes and hopefully identify novel sodium transporters from barley.

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses.

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Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol; Auesukaree, Choowong

2016-05-15

During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H(+)-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic

Acidification of the parasitophorous vacuole containing Toxoplasma gondii in the presence of hydroxyurea

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Cristiane S. Carvalho

2006-09-01

Full Text Available Toxoplasma gondii multiplies within parasitophorous vacuole that is not recognized by the primary no oxidative defense of host cells, mainly represented by the fusion with acidic organelles. Recent studies have already shown that hydroxyurea arrested the intracellular parasites leading to its destruction. In the present work we investigated the cellular mechanism involved in the destruction of intracellular Toxoplasma gondii. Fluorescent vital stains were used in order to observe possible acidification of parasitophorous vacuole-containing Toxoplasma gondii in presence of hydroxyurea. Vero cells infected with tachyzoites were treated with hydroxyurea for 12, 24 or 48 hours. Fluorescence, indicative of acidification, was observed in the parasitophorous vacuole when the cultures were incubated in presence of acridine orange. LysoTracker red was used in order to determine whether lysosomes were involved in the acidification process. An intense fluorescence was observed after 12 and 24 hours of incubation with hydroxyurea, achieving it is highly intensity after 48 hours of treatment. Ultrastructural cytochemistry for localization of the acid phosphatase lysosomal enzyme was realized. Treated infected cultures showed reaction product in vesicles fusing with vacuole or associated with intravacuolar parasites. These results suggest that fusion with lysosomes and acidification of parasitophorous vacuole leads to parasites destruction in the presence pf hydroxyurea.Toxoplasma gondii se multiplica dentro do vacúolo parasitóforo que não é reconhecido pela defesa primária não oxidativa de células hospedeiras: a fusão com organelas ácidas. Estudos anteriores mostraram que hidroxiuréia interrompeu a multiplicação dos parasitos intracelulares causando sua eliminação. No presente trabalho nós investigamos o mecanismo celular envolvido na destruição do Toxoplasma gondii intracelular. Marcadores vitais fluorescentes foram usados para observar a

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

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Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

2013-09-01

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The plant vacuolar Na+/H+ antiport.

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Barkla, B J; Apse, M P; Manolson, M F; Blumwald, E

1994-01-01

Salt stress imposes severe limitations on plant growth, however, the extent of growth reduction depends upon the soil salinity level and the plant species. One of the mechanisms employed by salt tolerant plants is the effective vacuolar compartmentalization of sodium. The sequestration of sodium into the vacuole occurs by the operation of a Na+/H+ antiport located at the tonoplast. Evidence for a plant vacuolar Na+/H+ antiport has been demonstrated in tissues, intact vacuoles and isolated tonoplast vesicles. In sugar beet cell suspensions, the activity of the vacuolar Na+/H+ antiport increased with increasing NaCl concentrations in the growth medium. This increased activity was correlated with the increased synthesis of a 170 kDa tonoplast polypeptide. In vivo labelling of tonoplast proteins showed the enhanced synthesis of the 170 kDa polypeptide not only upon exposure of the cells to salt, but also when the cells were grown in the presence of amiloride. Exposure of the cells to amiloride also resulted in increased vacuolar Na+/H+ antiport activity. Polyclonal antibodies raised against the 170 kDa polypeptide almost completely inhibited the antiport activity, suggesting the association of this protein with the plant vacuolar Na+/H+ antiport. Antibodies against the Na+/H+ antiport-associated polypeptide were used to screen a Beta lambda ZAP expression library. A partial clone of 1.65 kb was sequenced and found to encode a polypeptide with a putative transmembrane domain and a large hydrophilic C terminus. This clone showed no homology to any previously cloned gene at either the nucleic acid or the amino acid level.

Vacuolar iron transporter BnMEB2 is involved in enhancing iron tolerance of Brassica napus

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Wei Zhu

2016-09-01

Full Text Available Iron toxicity is a major nutrient disorder that severely affects crop development and yield. Vacuolar detoxification of metal stress is an important strategy for plants to survive and adapt to this adverse environment. Vacuolar iron transporter (VIT members are involved in this process and play essential roles in iron storage and transport. In this study, a rapeseed VIT gene BnMEB2 (BnaC07g30170D was identified. BnMEB2 is a homolog to Arabidopsis MEB2 (At5g24290 and acts as a detoxifier in vacuolar sequestration of divalent metal. Transient expression analysis revealed that BnMEB2 was localized to the vacuolar membrane. Q-PCR detection showed a high expression of BnMEB2 in mature (60-day-old leaves and could be obviously induced by exogenous iron stress in both roots and leaves. Over-expressed BnMEB2 in both Arabidopsis wild type and meb2 mutant seedlings resulted in greatly improved iron tolerability with no significant changes in the expression level of other vacuolar iron transporter genes. The mutant meb2 grew slowly and its root hair elongation was inhibited under high iron concentration condition while BnMEB2 over-expressed transgenic plants of the mutant restored the phenotypes with apparently higher iron storage in roots and dramatically increased iron content in the whole plant. Taken together, these results suggested that BnMEB2 was a VIT gene in rapeseed which was necessary for safe storage and vacuole detoxification function of excess iron to enhance the tolerance of iron toxicity. This research sheds light on a potentially new strategy for attenuating hazardous metal stress from environment and improving iron biofortification in Brassicaceae crops.

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

KAUST Repository

Wang, Zhen-Yu; Gehring, Christoph A; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

2014-01-01

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

KAUST Repository

Wang, Zhen-Yu

2014-11-21

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

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Summer R. Hayek

2014-01-01

Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

Sequential analysis of trans-SNARE formation in intracellular membrane fusion.

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Kannan Alpadi

2012-01-01

Full Text Available SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a, Q(b, and Q(c from target (t-SNAREs and one (R from the vesicular (v-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a SNARE, leaving behind a Q(bcR subcomplex. This subcomplex serves as an acceptor for a Q(a SNARE from the opposite membrane, leading to Q(a-Q(bcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bcR cis-complex and the formation of the Q(a-Q(bcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex. This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.

The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

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De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

2013-08-01

Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

Regulation of transport processes across the tonoplast membrane

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Oliver eTrentmann

2014-09-01

Full Text Available In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g. due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation.

A vacuolar iron transporter in tulip, TgVit1, is responsible for blue coloration in petal cells through iron accumulation.

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Momonoi, Kazumi; Yoshida, Kumi; Mano, Shoji; Takahashi, Hideyuki; Nakamori, Chihiro; Shoji, Kazuaki; Nitta, Akira; Nishimura, Mikio

2009-08-01

Blue color in flowers is due mainly to anthocyanins, and a considerable part of blue coloration can be attributed to metal-complexed anthocyanins. However, the mechanism of metal ion transport into vacuoles and subsequent flower color development has yet to be fully explored. Previously, we studied the mechanism of blue color development specifically at the bottom of the inner perianth in purple tulip petals of Tulipa gesneriana cv. Murasakizuisho. We found that differences in iron content were associated with the development of blue- and purple-colored cells. Here, we identify a vacuolar iron transporter in T. gesneriana (TgVit1), and characterize the localization and function of this transporter protein in tulip petals. The amino acid sequence of TgVit1 is 85% similar that of the Arabidopsis thaliana vacuolar iron transporter AtVIT1, and also showed similarity to the AtVIT1 homolog in yeast, Ca(2+)-sensitive cross-complementer 1 (CCC1). The gene TgVit1 was expressed exclusively in blue-colored epidermal cells, and protein levels increased with increasing mRNA expression and blue coloration. Transient expression experiments revealed that TgVit1 localizes to the vacuolar membrane, and is responsible for the development of the blue color in purple cells. Expression of TgVit1 in yeast rescued the growth defect of ccc1 mutant cells in the presence of high concentrations of FeSO(4). Our results indicate that TgVit1 plays an essential role in blue coloration as a vacuolar iron transporter in tulip petals. These results suggest a new role for involvement of a vacuolar iron transporter in blue flower color development.

AtHMA3, a P1B-ATPase allowing Cd/Zn/Co/Pb vacuolar storage in Arabidopsis.

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Morel, Mélanie; Crouzet, Jérôme; Gravot, Antoine; Auroy, Pascaline; Leonhardt, Nathalie; Vavasseur, Alain; Richaud, Pierre

2009-02-01

The Arabidopsis (Arabidopsis thaliana) Heavy Metal Associated3 (AtHMA3) protein belongs to the P1B-2 subgroup of the P-type ATPase family, which is involved in heavy metal transport. In a previous study, we have shown, using heterologous expression in the yeast Saccharomyces cerevisiae, that in the presence of toxic metals, AtHMA3 was able to phenotypically complement the cadmium/lead (Cd/Pb)-hypersensitive strain ycf1 but not the zinc (Zn)-hypersensitive strain zrc1. In this study, we demonstrate that AtHMA3 in planta is located in the vacuolar membrane, with a high expression level in guard cells, hydathodes, vascular tissues, and the root apex. Confocal imaging in the presence of the Zn/Cd fluorescent probe BTC-5N revealed that AtHMA3 participates in the vacuolar storage of Cd. A T-DNA insertional mutant was found more sensitive to Zn and Cd. Conversely, ectopic overexpression of AtHMA3 improved plant tolerance to Cd, cobalt, Pb, and Zn; Cd accumulation increased by about 2- to 3-fold in plants overexpressing AtHMA3 compared with wild-type plants. Thus, AtHMA3 likely plays a role in the detoxification of biological (Zn) and nonbiological (Cd, cobalt, and Pb) heavy metals by participating in their vacuolar sequestration, an original function for a P1B-2 ATPase in a multicellular eukaryote.

The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase.

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Namkoong, Sim; Lee, Kang Il; Lee, Jin I; Park, Rackhyun; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

2015-01-01

The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

Functional size of vacuolar H+ pumps: Estimates from radiation inactivation studies

International Nuclear Information System (INIS)

Sarafian, V.; Poole, R.J.

1991-01-01

The PPase and the ATPase from red beet (Beta vulgaris) vacuolar membranes were subjected to radiation inactivation by a 60 Co source in both the native tonoplast and detergent-solubilized states, in order to determine their target molecular sizes. Analysis of the residual phosphohydrolytic and proton transport activities, after exposure to varying doses of radiation, yielded exponential relationships between the activities and radiation doses. The deduced target molecular sizes for PPase activity in native and solubilized membranes were 125kD and 259kD respectively and 327kD for H + -transport. This suggests that the minimum number of subunits of 67kD for PPi hydrolysis is two in the native state and four after Triton X-100 solubilization. At least four subunits would be required for H + -translocation. Analysis of the ATPase inactivation patterns revealed target sizes of 384kD and 495kD for ATP hydrolysis in native and solubilized tonoplast respectively and 430kD for H + -transport. These results suggest that the minimum size for hydrolytic or transport functions is relatively constant for the ATPase

Miopatia vacuolar do lupus eritematoso

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José Antonio Levy

1962-06-01

Full Text Available Após considerações rápidas sôbre as miosites, particularmente sôbre a miopatia vacuolar do lúpus eritematoso, é relatado o caso de uma paciente no qual êsse diagnóstico pôde ser confirmado pelo exame histopatológico.

Targeting vacuolar H+-ATPases as a new strategy against cancer.

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Fais, Stefano; De Milito, Angelo; You, Haiyan; Qin, Wenxin

2007-11-15

Growing evidence suggests a key role of tumor acidic microenvironment in cancer development, progression, and metastasis. As a consequence, the need for compounds that specifically target the mechanism(s) responsible for the low pH of tumors is increasing. Among the key regulators of the tumor acidic microenvironment, vacuolar H(+)-ATPases (V-ATPases) play an important role. These proteins cover a number of functions in a variety of normal as well as tumor cells, in which they pump ions across the membranes. We discuss here some recent results showing that a molecular inhibition of V-ATPases by small interfering RNA in vivo as well as a pharmacologic inhibition through proton pump inhibitors led to tumor cytotoxicity and marked inhibition of human tumor growth in xenograft models. These results propose V-ATPases as a key target for new strategies in cancer treatment.

Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

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Helene Tournu

Full Text Available The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs. Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

The Arabidopsis vacuolar malate channel is a member of the ALMT family.

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Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

2007-12-01

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

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Shima, Jun; Ando, Akira; Takagi, Hiroshi

2008-03-01

Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance. (c) 2008 John Wiley & Sons, Ltd.

Bacterial pathogen manipulation of host membrane trafficking.

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Asrat, Seblewongel; de Jesús, Dennise A; Hempstead, Andrew D; Ramabhadran, Vinay; Isberg, Ralph R

2014-01-01

Pathogens use a vast number of strategies to alter host membrane dynamics. Targeting the host membrane machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Membrane manipulation promotes bacterial replication while suppressing host responses, allowing the bacterium to thrive in a hostile environment. This review provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack host membrane trafficking machinery. We start with mechanisms used by bacteria to alter the plasma membrane, delve into the hijacking of various vesicle trafficking pathways, and conclude by summarizing bacterial adaptation to host immune responses. Understanding bacterial manipulation of host membrane trafficking provides insights into bacterial pathogenesis and uncovers the molecular mechanisms behind various processes within a eukaryotic cell.

Vacuolar processing enzyme in plant programmed cell death

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Noriyuki eHatsugai

2015-04-01

Full Text Available Vacuolar processing enzyme (VPE is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an orthologue of animal asparaginyl endopeptidase (AEP/VPE/legumain. VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1.

Inorganic polyphosphate in the yeast Saccharomyces cerevisiae with a mutation disturbing the function of vacuolar ATPase.

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Tomaschevsky, A A; Ryasanova, L P; Kulakovskaya, T V; Kulaev, I S

2010-08-01

A mutation in the vma2 gene disturbing V-ATPase function in the yeast Saccharomyces cerevisiae results in a five- and threefold decrease in inorganic polyphosphate content in the stationary and active phases of growth on glucose, respectively. The average polyphosphate chain length in the mutant cells is decreased. The mutation does not prevent polyphosphate utilization during cultivation in a phosphate-deficient medium and recovery of its level on reinoculation in complete medium after phosphate deficiency. The content of short chain acid-soluble polyphosphates is recovered first. It is supposed that these polyphosphates are less dependent on the electrochemical gradient on the vacuolar membrane.

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

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

2000-06-01

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

Rapid Nuclear Exclusion of Hcm1 in Aging Saccharomyces cerevisiae Leads to Vacuolar Alkalization and Replicative Senescence

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Ata Ghavidel

2018-05-01

Full Text Available The yeast, Saccharomyces cerevisiae, like other higher eukaryotes, undergo a finite number of cell divisions before exiting the cell cycle due to the effects of aging. Here, we show that yeast aging begins with the nuclear exclusion of Hcm1 in young cells, resulting in loss of acidic vacuoles. Autophagy is required for healthy aging in yeast, with proteins targeted for turnover by autophagy directed to the vacuole. Consistent with this, vacuolar acidity is necessary for vacuolar function and yeast longevity. Using yeast genetics and immunofluorescence microscopy, we confirm that vacuolar acidity plays a critical role in cell health and lifespan, and is potentially maintained by a series of Forkhead Box (Fox transcription factors. An interconnected transcriptional network involving the Fox proteins (Fkh1, Fkh2 and Hcm1 are required for transcription of v-ATPase subunits and vacuolar acidity. As cells age, Hcm1 is rapidly excluded from the nucleus in young cells, blocking the expression of Hcm1 targets (Fkh1 and Fkh2, leading to loss of v-ATPase gene expression, reduced vacuolar acidification, increased α-syn-GFP vacuolar accumulation, and finally, diminished replicative lifespan (RLS. Loss of vacuolar acidity occurs about the same time as Hcm1 nuclear exclusion and is conserved; we have recently demonstrated that lysosomal alkalization similarly contributes to aging in C. elegans following a transition from progeny producing to post-reproductive life. Our data points to a molecular mechanism regulating vacuolar acidity that signals the end of RLS when acidification is lost.

Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).

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Nakamura, Atsuko; Fukuda, Atsunori; Sakai, Shingo; Tanaka, Yoshiyuki

2006-01-01

We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen Cryptococcus neoformans.

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Cho, Minsu; Hu, Guanggan; Caza, Mélissa; Horianopoulos, Linda C; Kronstad, James W; Jung, Won Hee

2018-01-01

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding[OPEN

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Vincent, Thomas R.; Avramova, Marieta; Canham, James; Higgins, Peter; Bilkey, Natasha; Mugford, Sam T.; Pitino, Marco; Toyota, Masatsugu

2017-01-01

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction. PMID:28559475

Cryo-EM studies of the structure and dynamics of vacuolar-type ATPases

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Mazhab-Jafari, Mohammad T.; Rubinstein, John L.

2016-01-01

Electron cryomicroscopy (cryo-EM) has significantly advanced our understanding of molecular structure in biology. Recent innovations in both hardware and software have made cryo-EM a viable alternative for targets that are not amenable to x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Cryo-EM has even become the method of choice in some situations where x-ray crystallography and NMR spectroscopy are possible but where cryo-EM can determine structures at higher resolution or with less time or effort. Rotary adenosine triphosphatases (ATPases) are crucial to the maintenance of cellular homeostasis. These enzymes couple the synthesis or hydrolysis of adenosine triphosphate to the use or production of a transmembrane electrochemical ion gradient, respectively. However, the membrane-embedded nature and conformational heterogeneity of intact rotary ATPases have prevented their high-resolution structural analysis to date. Recent application of cryo-EM methods to the different types of rotary ATPase has led to sudden advances in understanding the structure and function of these enzymes, revealing significant conformational heterogeneity and characteristic transmembrane α helices that are highly tilted with respect to the membrane. In this Review, we will discuss what has been learned recently about rotary ATPase structure and function, with a particular focus on the vacuolar-type ATPases. PMID:27532044

Regulation of Vacuolar pH in Citrus limon

Energy Technology Data Exchange (ETDEWEB)

Lincoln Taiz

2005-06-22

The primary objective of this grant was to characterize the vacuolar V-ATPase of lemon fruits. Lemon fruit vacuoles have an internal pH of about 2.5. Since a typical plant vacuole has a luminal pH of around 5.5, the lemon fruit V-APTase must have special properties which allow it to acidify the lumen to such a low pH: (1) it might have a different structure; (2) it might have a different H{sup +}/ATP stoichiometry; and (3) it might be regulated differently. During the course of the investigations (which began in 1996) they characterized these aspects of the V-ATPases of both lemon fruits and lime fruits. They examined lime fruits because of the availability of both acidic limes with a low vacuolar pH and sweet limes, which have a much higher vacuolar pH. The existence of two types of lime fruits allowed a comparison of the V-ATPases of the two varieties. In this report they are including two publications from 1996 and 1997 as background for the later publications. A review article with Heven Sze on V-ATPase nomenclature was also generated during the funding period. In addition to the studies on citrus fruit vacuoles, they also initiated studies in two new areas: polar auxin transport and the regulation of stomatal opening by UV-B irradiation. These studies were intended to serve as a basis of future separate grants, but the proposals they submitted on these topics were not funded.

Characterization of the vacuolar H sup + -ATPase of higher plants

Energy Technology Data Exchange (ETDEWEB)

Manolson, M F

1988-01-01

The tonoplast H{sup +}-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides. Kinetic analysis of ({alpha}-{sup 32}P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ({sup 14}C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H{sup +}-ATPase is a multimer composed of at least three polypeptides. Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H{sup +}-ATPase of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F{sub 0}F{sub 1} and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

NMR spectroscopic studies of membrane-bound biological systems

International Nuclear Information System (INIS)

Hohlweg, W.

2013-01-01

In the course of this thesis, biological NMR spectroscopy was employed in studying membrane-bound peptides and proteins, for which structural information is still comparatively hard to obtain. Initial work focused on various model peptides bound to membrane-mimicking micelles, studying the protonation state of arginine in a membrane environment. Strong evidence for a cation-π complex was found in TM7, a peptide which forms the seventh transmembrane helix of subunit a of the vacuolar-type H+-ATPase (V-ATPase). V-ATPase is a physiologically highly relevant proton pump, which is present in intracellular membranes of all eukaryotic organisms, as well as the plasma membrane of several specialized cells. Loss of functional V-ATPase is associated with human diseases such as osteopetrosis, distal renal tubular acidosis or the spreading of cancer. V-ATPase is considered a potential drug target in the treatment of osteoporosis and cancer, or in the development of novel contraceptives. Results from NMR solution structure determination, NMR titration experiments, paramagnetic relaxation enhancement experiments and tryptophan fluorescence spectroscopy confirm the existence of a buried cation-? complex formed between arginine residue R735, which is essential for proton transport, and neighbouring tryptophan and tyrosine residues. In vivo experiments in the yeast Saccharomyces cerevisiae using selective growth tests and fluorescence microscopy showed that formation of the cation-π complex is essential for V-ATPase function. Deletion of both aromatic residues, as well as only the one tryptophan residue leads to growth defects and inability to maintain vacuolar pH homeostasis. These findings shine new light on the still elusive mechanism of proton transport in V-ATPase, and show that arginine R735 may be directly involved in proton transfer across the membrane. (author) [de

Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

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Aurélie Fougère

2016-11-01

Full Text Available Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC. P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

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Hemenway, C S; Dolinski, K; Cardenas, M E; Hiller, M A; Jones, E W; Heitman, J

1995-11-01

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

Manipulation of the Host Cell Membrane during Plasmodium Liver Stage Egress

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Paul-Christian Burda

2017-04-01

Full Text Available A crucial step in the life cycle of Plasmodium parasites is the transition from the liver stage to the blood stage. Hepatocyte-derived merozoites reach the blood vessels of the liver inside host cell-derived vesicles called merosomes. The molecular basis of merosome formation is only partially understood. Here we show that Plasmodium berghei liver stage merozoites, upon rupture of the parasitophorous vacuole membrane, destabilize the host cell membrane (HCM and induce separation of the host cell actin cytoskeleton from the HCM. At the same time, the phospholipid and protein composition of the HCM appears to be substantially altered. This includes the loss of a phosphatidylinositol 4,5-bisphosphate (PIP2 reporter and the PIP2-dependent actin-plasma membrane linker ezrin from the HCM. Furthermore, transmembrane domain-containing proteins and palmitoylated and myristoylated proteins, as well as glycosylphosphatidylinositol-anchored proteins, lose their HCM localization. Collectively, these findings provide an explanation of HCM destabilization during Plasmodium liver stage egress and thereby contribute to our understanding of the molecular mechanisms that lead to merosome formation.

Tonoplast- and plasma membrane-localized aquaporin-family transporters in blue hydrangea sepals of aluminum hyperaccumulating plant.

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Takashi Negishi

Full Text Available Hydrangea (Hydrangea macrophylla is tolerant of acidic soils in which toxicity generally arises from the presence of the soluble aluminum (Al ion. When hydrangea is cultivated in acidic soil, its resulting blue sepal color is caused by the Al complex formation of anthocyanin. The concentration of vacuolar Al in blue sepal cells can reach levels in excess of approximately 15 mM, suggesting the existence of an Al-transport and/or storage system. However, until now, no Al transporter has been identified in Al hyperaccumulating plants, animals or microorganisms. To identify the transporter being responsible for Al hyperaccumulation, we prepared a cDNA library from blue sepals according to the sepal maturation stage, and then selected candidate genes using a microarray analysis and an in silico study. Here, we identified the vacuolar and plasma membrane-localized Al transporters genes vacuolar Al transporter (VALT and plasma membrane Al transporter 1 (PALT1, respectively, which are both members of the aquaporin family. The localization of each protein was confirmed by the transient co-expression of the genes. Reverse transcription-PCR and immunoblotting results indicated that VALT and PALT1 are highly expressed in sepal tissue. The overexpression of VALT and PALT1 in Arabidopsis thaliana conferred Al-tolerance and Al-sensitivity, respectively.

RNAi-directed downregulation of vacuolar H(+ -ATPase subunit a results in enhanced stomatal aperture and density in rice.

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Huiying Zhang

Full Text Available Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L. vacuolar H(+-ATPase subunit A (OsVHA-A gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+-ATPase activity and an enhancement of plasma membrane H(+-ATPase activity, thereby increasing the concentrations of extracellular H(+ and intracellular K(+ and Na(+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+-ATPase 3 and downregulation of CAM1 (calmodulin 1, CAM3 (calmodulin 3 and YDA1 (YODA, a MAPKK gene. Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

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Stransky, Laura A.; Forgac, Michael

2015-01-01

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

Cytokine expression of macrophages in HIV-1-associated vacuolar myelopathy.

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Tyor, W R; Glass, J D; Baumrind, N; McArthur, J C; Griffin, J W; Becker, P S; Griffin, D E

1993-05-01

Macrophages are frequently present within the periaxonal and intramyelinic vacuoles that are located primarily in the posterior and lateral funiculi of the thoracic spinal cord in HIV-associated vacuolar myelopathy. But the role of these macrophages in the formation of the vacuoles is unclear. One hypothesis is that cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF)-alpha, are produced locally by macrophages and have toxic effects on myelin or oligodendrocytes. The resulting myelin damage eventually culminates in the removal of myelin by macrophages and vacuole formation. We studied thoracic spinal cord specimens taken at autopsy from HIV-positive (+) and HIV-negative individuals. The predominant mononuclear cells present in HIV+ spinal cords are macrophages. They are located primarily in the posterior and lateral funiculi regardless of the presence or absence of vacuolar myelopathy. Macrophages and microglia are more frequent in HIV+ than HIV-negative individuals and these cells frequently stain for class I and class II antigens, IL-1, and TNF-alpha. Activated macrophages positive for IL-1 and TNF-alpha are great increased in the posterior and lateral funiculi of HIV+ individuals with and without vacuolar myelopathy, suggesting they are present prior to the development of vacuoles. Cytokines, such as TNF-alpha, may be toxic for myelin or oligodendrocytes, leading to myelin damage and removal by macrophages and vacuole formation.

Multiple pathways for vacuolar sorting of yeast proteinase A

DEFF Research Database (Denmark)

Westphal, V; Marcusson, E G; Winther, Jakob R.

1996-01-01

The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide...

Covalent structures of potato tuber lipases (patatins) and implications for vacuolar import

DEFF Research Database (Denmark)

Welinder, Karen Gjesing; Jørgensen, Malene

that the path is via the Golgi apparatus. However, the vacuolar targeting signal has never been identified for this storage and defence protein, which amounts to 25-40% of tuber protein. We propose that a six-residue ct-propeptide, -ANKASY-COO- composes this signal. The crystallographic structure...... the Danish Research Council for Technology and Production, and grant 2052-03-0022 from the Danish Research Agency.   Reference Welinder KG, Jørgensen M (2009) Covalent structures of potato tuber lipases (patatins) and implications for vacuolar import. J. Biol. Chem., Feb 2009; doi:10.1074/jbc.M809674200....

RNAi-based silencing of genes encoding the vacuolar- ATPase ...

African Journals Online (AJOL)

RNAi-based silencing of genes encoding the vacuolar- ATPase subunits a and c in pink bollworm (Pectinophora gossypiella). Ahmed M. A. Mohammed. Abstract. RNA interference is a post- transcriptional gene regulation mechanism that is predominantly found in eukaryotic organisms. RNAi demonstrated a successful ...

The diverse and dynamic nature of Leishmania parasitophorous vacuoles studied by multidimensional imaging.

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Fernando Real

Full Text Available An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i hosting amastigotes of either L. major or L. amazonensis and ii loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms.

The Arabidopsis cax3 mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H+-ATPase activity.

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Zhao, Jian; Barkla, Bronwyn J; Marshall, Joy; Pittman, Jon K; Hirschi, Kendal D

2008-02-01

Perturbing CAX1, an Arabidopsis vacuolar H+/Ca2+ antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among cax1 and cax3 including an increased sensitivity to both abscisic acid (ABA) and sugar during germination, and an increased tolerance to ethylene during early seedling development. We have also identified phenotypes unique to cax3, namely salt, lithium and low pH sensitivity. We used biochemical measurements to ascribe these cax3 sensitivities to a reduction in vacuolar H+/Ca2+ transport during salt stress and decreased plasma membrane H+-ATPase activity. These findings catalog an array of CAX phenotypes and assign a specific role for CAX3 in response to salt tolerance.

Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

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Ronzone, Erik; Paumet, Fabienne

2013-01-01

Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.

Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

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Erik Ronzone

Full Text Available Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.

Appropriate vacuolar acidification in Saccharomyces cerevisiae is associated with efficient high sugar fermentation.

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Nguyen, Trung D; Walker, Michelle E; Gardner, Jennifer M; Jiranek, Vladimir

2018-04-01

Vacuolar acidification serves as a homeostatic mechanism to regulate intracellular pH, ion and chemical balance, as well as trafficking and recycling of proteins and nutrients, critical for normal cellular function. This study reports on the importance of vacuole acidification during wine-like fermentation. Ninety-three mutants (homozygous deletions in lab yeast strain, BY4743), which result in protracted fermentation when grown in a chemically defined grape juice with 200 g L -1 sugar (pH 3.5), were examined to determine whether fermentation protraction was in part due to a dysfunction in vacuolar acidification (VA) during the early stages of fermentation, and whether VA was responsive to the initial sugar concentration in the medium. Cells after 24 h growth were dual-labelled with propidium iodide and vacuolar specific probe 6-carboxyfluorescein diacetate (6-CFDA) and examined with a FACS analyser for viability and impaired VA, respectively. Twenty mutants showed a greater than two-fold increase in fluorescence intensity; the experimental indicator for vacuolar dysfunction; 10 of which have not been previously annotated to this process. With the exception of Δhog1, Δpbs2 and Δvph1 mutants, where dysfunction was directly related to osmolality; the remainder exhibited increased CF-fluorescence, independent of sugar concentration at 20 g L -1 or 200 g L -1 . These findings offer insight to the importance of VA to cell growth in high sugar media. Copyright © 2017 Elsevier Ltd. All rights reserved.

Membrane proteins involved in transport, vesicle traffic and Ca(2+) signaling increase in beetroots grown in saline soils.

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Lino, Bárbara; Chagolla, Alicia; E González de la Vara, Luis

2016-07-01

By separating plasma membrane proteins according to their hydropathy from beetroots grown in saline soils, several proteins probably involved in salt tolerance were identified by mass spectrometry. Beetroots, as a salt-tolerant crop, have developed mechanisms to cope with stresses associated with saline soils. To observe which plasma membrane (PM) proteins were more abundant in beet roots grown in saline soils, beet root plants were irrigated with water or 0.2 M NaCl. PM-enriched membrane preparations were obtained from these plants, and their proteins were separated according to their hydropathy by serial phase partitioning with Triton X-114. Some proteins whose abundance increased visibly in membranes from salt-grown beetroots were identified by mass spectrometry. Among them, there was a V-type H(+)-ATPase (probably from contaminating vacuolar membranes), which increased with salt at all stages of beetroots' development. Proteins involved in solute transport (an H(+)-transporting PPase and annexins), vesicle traffic (clathrin and synaptotagmins), signal perception and transduction (protein kinases and phospholipases, mostly involved in calcium signaling) and metabolism, appeared to increase in salt-grown beetroot PM-enriched membranes. These results suggest that PM and vacuolar proteins involved in transport, metabolism and signal transduction increase in beet roots adapted to saline soils. In addition, these results show that serial phase partitioning with Triton X-114 is a useful method to separate membrane proteins for their identification by mass spectrometry.

Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy.

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Spugnini, Enrico P; Citro, Gennaro; Fais, Stefano

2010-05-08

The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy

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Fais Stefano

2010-05-01

Full Text Available Abstract The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

International Nuclear Information System (INIS)

Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

2009-01-01

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP 2 and PIP 3 to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

The Amborella vacuolar processing enzyme family

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Valérie ePoncet

2015-08-01

Full Text Available Most vacuolar proteins are synthesized on rough endoplasmic reticulum as proprotein precursors and then transported to the vacuoles, where they are converted into their respective mature forms by vacuolar processing enzymes (VPEs. In the case of the seed storage proteins, this process is of major importance, as it conditions the establishment of vigorous seedlings. Toward the goal of identifying proteome signatures that could be associated with the origin and early diversification of angiosperms, we previously characterized the 11S-legumin-type of seed storage proteins from Amborella trichopoda, a rainforest shrub endemic to New Caledonia that is also the probable sister to all other angiosperms (Amborella Genome Project, 2013. In the present study, proteomic and genomic approaches were used to characterize the VPE family in this species. Three genes were found to encode VPEs in the Amborella’s genome. Phylogenetic analyses showed that the Amborella sequences grouped within two major clades of angiosperm VPEs, indicating that the duplication that generated the ancestors of these clades occurred before the most recent common ancestor of living angiosperms. A further important duplication within the VPE family appears to have occurred in common ancestor of the core eudicots, while many more recent duplications have also occurred in specific taxa, including both Arabidopsis thaliana and Amborella. An analysis of natural genetic variation for each of the three Amborella VPE genes revealed the absence of selective forces acting on intronic and exonic single-nucleotide polymorphisms among several natural Amborella populations of in New Caledonia.

Transport pathways in the malaria-infected erythrocyte: characterization and their use as potential targets for chemotherapy

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Hagai Ginsburg

1994-01-01

Full Text Available The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste product, are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. When the new pathways are quantitatively unimportant, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to effectively inhibit the new pathways and consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels which are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters which respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidences. No specific inhibitors were found as yet that would efficiently inhibit transport through the channels or the duct.

Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens

DEFF Research Database (Denmark)

van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.

1992-01-01

The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. Co...

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

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Bárbara Hissa

Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of

Inward cholesterol gradient of the membrane system in P. falciparum-infected erythrocytes involves a dilution effect from parasite-produced lipids

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Fuyuki Tokumasu

2014-05-01

Full Text Available Plasmodium falciparum (Pf infection remodels the human erythrocyte with new membrane systems, including a modified host erythrocyte membrane (EM, a parasitophorous vacuole membrane (PVM, a tubulovesicular network (TVN, and Maurer's clefts (MC. Here we report on the relative cholesterol contents of these membranes in parasitized normal (HbAA and hemoglobin S-containing (HbAS, HbAS erythrocytes. Results from fluorescence lifetime imaging microscopy (FLIM experiments with a cholesterol-sensitive fluorophore show that membrane cholesterol levels in parasitized erythrocytes (pRBC decrease inwardly from the EM, to the MC/TVN, to the PVM, and finally to the parasite membrane (PM. Cholesterol depletion of pRBC by methyl-β-cyclodextrin treatment caused a collapse of this gradient. Lipid and cholesterol exchange data suggest that the cholesterol gradient involves a dilution effect from non-sterol lipids produced by the parasite. FLIM signals from the PVM or PM showed little or no difference between parasitized HbAA vs HbS-containing erythrocytes that differed in lipid content, suggesting that malaria parasites may regulate the cholesterol contents of the PVM and PM independently of levels in the host cell membrane. Cholesterol levels may affect raft structures and the membrane trafficking and sorting functions that support Pf survival in HbAA, HbAS and HbSS erythrocytes.

Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

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Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

2014-01-01

The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

International Nuclear Information System (INIS)

Polatnick, J.; Wool, S.H.

1982-01-01

Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [ 3 H] uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity. (Author)

Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

Energy Technology Data Exchange (ETDEWEB)

Polatnick, J.; Wool, S.H. (United States Department of Agriculture, Science and Education, Greenport, New York (USA). Agricultural Research, Plum Island Animal Disease Center)

1982-01-01

Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated (/sup 3/H) uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity.

Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET

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Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael

2010-02-01

We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

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Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

2013-09-15

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

International Nuclear Information System (INIS)

Kohio, Hinissan P.; Adamson, Amy L.

2013-01-01

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier.

Science.gov (United States)

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A; Traub, Michaela; Martinoia, Enrico; Neuhaus, H Ekkehard

2003-09-16

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter.

Modeling the vacuolar storage of malate shed lights on pre- and post-harvest fruit acidity.

Science.gov (United States)

Etienne, Audrey; Génard, Michel; Lobit, Philippe; Bugaud, Christophe

2014-11-18

Malate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. Several studies suggest that malate accumulation in fruit cells is controlled at the level of vacuolar storage. However, the regulation of vacuolar malate storage throughout fruit development, and the origins of the phenotypic variability of the malate concentration within fruit species remain to be clarified. In the present study, we adapted the mechanistic model of vacuolar storage proposed by Lobit et al. in order to study the accumulation of malate in pre and postharvest fruits. The main adaptation concerned the variation of the free energy of ATP hydrolysis during fruit development. Banana fruit was taken as a reference because it has the particularity of having separate growth and post-harvest ripening stages, during which malate concentration undergoes substantial changes. Moreover, the concentration of malate in banana pulp varies greatly among cultivars which make possible to use the model as a tool to analyze the genotypic variability. The model was calibrated and validated using data sets from three cultivars with contrasting malate accumulation, grown under different fruit loads and potassium supplies, and harvested at different stages. The model predicted the pre and post-harvest dynamics of malate concentration with fairly good accuracy for the three cultivars (mean RRMSE = 0.25-0.42). The sensitivity of the model to parameters and input variables was analyzed. According to the model, vacuolar composition, in particular potassium and organic acid concentrations, had an important effect on malate accumulation. The model suggested that rising temperatures depressed malate accumulation. The model also helped distinguish differences in malate concentration among the three cultivars and between the pre and post-harvest stages by highlighting the probable importance of proton pump activity and particularly of the free

Intense pseudotransport of a cationic drug mediated by vacuolar ATPase: Procainamide-induced autophagic cell vacuolization

International Nuclear Information System (INIS)

Morissette, Guillaume; Lodge, Robert; Marceau, Francois

2008-01-01

Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent K M 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (≥ 24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking

Evolutionary divergence of plant borate exporters and critical amino acid residues for the polar localization and boron-dependent vacuolar sorting of AtBOR1

KAUST Repository

Wakuta, Shinji

2015-01-24

Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Transient anterior subcapsular vacuolar change of the crystalline lens in patients after posterior chamber phakic intraocular lens implantation.

Science.gov (United States)

Chung, Jin Kwon; Shin, Jin Hee; Lee, Sung Jin

2013-10-25

We present two cases of transient vacuolar changes in the anterior subcapsular space of the crystalline lens in patients after posterior chamber phakic intraocular lens implantation. Implantable collamer lenses (ICL) were implanted in healthy myopic patients. Vacuolar changes developed just after the irrigating procedure through the narrow space between the ICL and the crystalline lens. Slit-lamp examinations and spectral domain optical coherence tomography showed bleb-like lesions in the anterior subcapsular space of one eye in each case, though the lesions gradually improved without visual deterioration. Consequently, the lesions turned into a few anterior subcapsular small faint opacities. Direct irrigation of the narrow space confined by the ICL and the crystalline lens is at risk for the development of vacuolar changes in the crystalline lens. The observed spontaneous reversal indicates that surgeons should not rush to surgical intervention but rather opt for close follow over several weeks.

Effect of external pH on the cytoplasmic and vacuolar pHs in Mung bean root-tip cells

International Nuclear Information System (INIS)

Torimitsu, Keiichi; Yazaki, Yoshiaki; Nagasuka, Kinuyo; Ohta, Eiji; Sakata, Makoto

1984-01-01

The effect of the external pH on the intracellular pH in mung bean (Vigna mungo (L.) Hepper) root-tip cells was investigated with the 31 P nuclear magnetic resonance (NMR) method. The 31 P NMR spectra showed three peaks caused by cytoplasmic G-6-P, cytoplasmic Psub(i) and vacuolar Psub(i). The cytoplasmic and vacuolar pHs could be determined by comparing the Psub(i) chemical shifts with the titration curve. When the external pH was changed over a range from pH 3 to 10, the cytoplasmic pH showed smaller changes than the vacuolar pH, suggesting that the former is regulated more strictly than the latter. The H + -ATPase inhibitor, DCCD, caused the breakdown of the mechanism that regulates the intracellular pH. H + -ATPase appears to have an important part in the regulation of the intracellular pH. (author)

Leishmania hijacking of the macrophage intracellular compartments.

Science.gov (United States)

Liévin-Le Moal, Vanessa; Loiseau, Philippe M

2016-02-01

Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses. © 2015 FEBS.

The ETRAMP family member SEP2 is expressed throughout Plasmodium berghei life cycle and is released during sporozoite gliding motility.

Science.gov (United States)

Currà, Chiara; Di Luca, Marco; Picci, Leonardo; de Sousa Silva Gomes dos Santos, Carina; Siden-Kiamos, Inga; Pace, Tomasino; Ponzi, Marta

2013-01-01

The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.

Vacuolar myelinopathy in waterfowl from a North Carolina impoundment

Science.gov (United States)

Augspurger, T.; Fischer, John R.; Thomas, Nancy; Sileo, L.; Brannian, Roger E.; Miller, Kimberli J.; Rocke, Tonie E.

2003-01-01

Vacuolar myelinopathy was confirmed by light and electron microscopic examination of mallards (Anas platyrhynchos), ring-necked ducks (Aythya collaris), and buffleheads (Bucephala albeola) collected during an epizootic at Lake Surf in central North Carolina (USA) between November 1998 and February 1999. Clinical signs of affected birds were consistent with central nervous system impairment of motor function (incoordination, abnormal movement and posture, weakness, paralysis). This is the first report of this disease in wild waterfowl (Anseriformes).Aug

Covalent structures of potato tuber lipases (patatins) and implications for vacuolar import

DEFF Research Database (Denmark)

Welinder, Karen Gjesing; Jørgensen, Malene

2009-01-01

patatin (Rydel, T. J., Williams, J. M., Krieger, E., Moshiri, F., Stallings, W. C., Brown, S. M., Pershing, J. C., Prucell, J. P., and Alibhai, M. F. (2003) Biochemistry 42, 6696-6708), which included this propeptide thus, for the first time, shows the structure of a putative ligand of the vacuolar...

The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion.

Science.gov (United States)

Schulte, Kathrin; Pawlowski, Nikolaus; Faelber, Katja; Fröhlich, Chris; Howard, Jonathan; Daumke, Oliver

2016-03-02

The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action.

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

International Nuclear Information System (INIS)

Milgrom, Elena M.; Milgrom, Yakov M.

2012-01-01

Highlights: ► MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. ► V-ATPase activity saturation with MgATP is not sufficient for complete protection. ► The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K m values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

Science.gov (United States)

Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

2016-02-01

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Highly Efficient Single-Step Enrichment of Low Abundance Phosphopeptides from Plant Membrane Preparations

Directory of Open Access Journals (Sweden)

Xu Na Wu

2017-09-01

Full Text Available Mass spectrometry (MS-based large scale phosphoproteomics has facilitated the investigation of plant phosphorylation dynamics on a system-wide scale. However, generating large scale data sets for membrane phosphoproteins usually requires fractionation of samples and extended hands-on laboratory time. To overcome these limitations, we developed “ShortPhos,” an efficient and simple phosphoproteomics protocol optimized for research on plant membrane proteins. The optimized workflow allows fast and efficient identification and quantification of phosphopeptides, even from small amounts of starting plant materials. “ShortPhos” can produce label-free datasets with a high quantitative reproducibility. In addition, the “ShortPhos” protocol recovered more phosphorylation sites from membrane proteins, especially plasma membrane and vacuolar proteins, when compared to our previous workflow and other membrane-based data in the PhosPhAt 4.0 database. We applied “ShortPhos” to study kinase-substrate relationships within a nitrate-induction experiment on Arabidopsis roots. The “ShortPhos” identified significantly more known kinase-substrate relationships compared to previous phosphoproteomics workflows, producing new insights into nitrate-induced signaling pathways.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells

Science.gov (United States)

Mitchell, Gabriel

2017-01-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called “viable but non-culturable” state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy. PMID:29190284

Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species.

NARCIS (Netherlands)

Silvie, O.; Greco, C.; Franetich, J.F.; Dubart-Kupperschmitt, A.; Hannoun, L.; Gemert, G.J.A. van; Sauerwein, R.W.; Levy, S.; Boucheix, C.; Rubinstein, E.; Mazier, D.

2006-01-01

Plasmodium sporozoites can enter host cells by two distinct pathways, either through disruption of the plasma membrane followed by parasite transmigration through cells, or by formation of a parasitophorous vacuole (PV) where the parasite further differentiates into a replicative exo-erythrocytic

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

Science.gov (United States)

Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

2012-01-01

PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

Vacuolar invertase gene silencing in potato (Solanum tuberosum L. improves processing quality by decreasing the frequency of sugar-end defects.

Directory of Open Access Journals (Sweden)

Xiaobiao Zhu

Full Text Available Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.

Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme

International Nuclear Information System (INIS)

Kasho, V.N.; Boyer, P.D.

1989-01-01

Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F 1 , F 0 -ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F 1 ,F 0 -ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and P i as medium ATP concentration was lowered was determined by 18 O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the P i formed as ATP concentration is decreased to the micromolar range. The F 1 ,F 0 -ATPase from neurospora mitochondria showed an event more pronounced modulation, similar to that of other F 1 -type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F 1 ,F 0 -ATPases

Macropinosomes are Key Players in Early Shigella Invasion and Vacuolar Escape in Epithelial Cells.

Directory of Open Access Journals (Sweden)

Allon Weiner

2016-05-01

Full Text Available Intracellular pathogens include all viruses, many bacteria and parasites capable of invading and surviving within host cells. Key to survival is the subversion of host cell pathways by the pathogen for the purpose of propagation and evading the immune system. The intracellular bacterium Shigella flexneri, the causative agent of bacillary dysentery, invades host cells in a vacuole that is subsequently ruptured to allow growth of the pathogen within the host cytoplasm. S. flexneri invasion has been classically described as a macropinocytosis-like process, however the underlying details and the role of macropinosomes in the intracellular bacterial lifestyle have remained elusive. We applied dynamic imaging and advanced large volume correlative light electron microscopy (CLEM to study the highly transient events of S. flexneri's early invasion into host epithelial cells and elucidate some of its fundamental features. First, we demonstrate a clear distinction between two compartments formed during the first step of invasion: the bacterial containing vacuole and surrounding macropinosomes, often considered identical. Next, we report a functional link between macropinosomes and the process of vacuolar rupture, demonstrating that rupture timing is dependent on the availability of macropinosomes as well as the activity of the small GTPase Rab11 recruited directly to macropinosomes. We go on to reveal that the bacterial containing vacuole and macropinosomes come into direct contact at the onset of vacuolar rupture. Finally, we demonstrate that S. flexneri does not subvert pre-existing host endocytic vesicles during the invasion steps leading to vacuolar rupture, and propose that macropinosomes are the major compartment involved in these events. These results provide the basis for a new model of the early steps of S. flexneri epithelial cell invasion, establishing a different view of the enigmatic process of cytoplasmic access by invasive bacterial

The potential of fluorinated surfactants in membrane biochemistry.

Science.gov (United States)

Shepherd, F H; Holzenburg, A

1995-01-01

Detergents are important reagents in membrane biochemistry. Since each membrane system studied places different demands on the detergent in terms of desirous physicochemical properties, detergents new to biochemistry must continuously be sought. Ammonium perfluorooctanoate (APFO) was investigated, as representative of fluorinated surfactants, in terms of its suitability as a "biological detergent." It did not interfere with the Markwell modification of the Lowry procedure at detergent concentrations of up to 2% (w/v). Critical micellization concentration (cmc) values (0.013-0.0275 M) for this detergent were determined in a number of buffers of biological interest. It was demonstrated that the detergent can be removed by dialysis, albeit slowly. This slow removal may be particularly useful for reconstitution/crystallization studies. Solubilization studies on several membrane systems containing the proteins listed (the major protein of the membrane sector of the vacuolar H(+)-ATPase (16 kDa protein); photosystem II; equine herpes virus (EHV) envelope proteins) indicate that it is a potent solubilizing agent, likely to enhance the yield in cases where solubilization has already been demonstrated, and, in other cases, to solubilize proteins formerly recalcitrant to solubilization. The removal of APFO from solubilized 16-kDa protein by means of Extracti-Gel D resin as a means of exchanging detergents quickly and with a minimum requirement for second detergent was investigated.

THE TONOPLAST TRANSPORT SYSTEMS OF PLANT VACUOLES AND THEIR POTENTIAL APPLICATION IN BIOTECHNOLOGY

Directory of Open Access Journals (Sweden)

S. V. Isayenkov

2013-06-01

Full Text Available The pivotal role of plant vacuoles in plant survival was discussed in the review. Particularly, the providing of cellular turgor, accumulation of inorganic osmolytes and nutrients are the primary tasks of these cellular organelles. The main mechanisms of tonoplast transport systems were described. The known transport pathways of minerals, heavy metals, vitamins and other organic compounds were classified and outlined. The main systems of membrane vacuolar transport were reviewed. The outline of the physiological functions and features of vacuolar membrane transport proteins were performed. The physiological role of transport of minerals, nutrients and other compounds into vacuoles were discussed. This article reviews the main types of plant vacuoles and their functional role in plant cell. Current state and progress in vacuolar transport research was outlined. The examples of application for rinciples and mechanisms of vacuolar membrane transport in plant biotechnology were iven. The perspectives and approaches in plant and food biotechnology concerning transport and physiology of vacuoles are discussed.

Vacuolar processing enzyme: an executor of plant cell death.

Science.gov (United States)

Hara-Nishimura, Ikuko; Hatsugai, Noriyuki; Nakaune, Satoru; Kuroyanagi, Miwa; Nishimura, Mikio

2005-08-01

Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

Shigella subverts the host recycling compartment to rupture its vacuole.

Science.gov (United States)

Mellouk, Nora; Weiner, Allon; Aulner, Nathalie; Schmitt, Christine; Elbaum, Michael; Shorte, Spencer L; Danckaert, Anne; Enninga, Jost

2014-10-08

Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion. Copyright © 2014 Elsevier Inc. All rights reserved.

Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

Science.gov (United States)

Pottosin, Igor; Bonales-Alatorre, Edgar; Shabala, Sergey

2014-11-03

Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Enhanced separation of membranes during free flow zonal electrophoresis in plants.

Science.gov (United States)

Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

2007-07-15

Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FFZE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201-213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface.

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

Science.gov (United States)

Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Grégoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

2018-05-22

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H + -ATPase (V-ATPase) and H + -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H + -ATPase (V-ATPase) and H + -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

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Chengliang Zhang

Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

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Annika Müller-Lucks

Full Text Available Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD, proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

Cryptosporidia: Epicellular parasites embraced by the host cell membrane

Czech Academy of Sciences Publication Activity Database

Valigurová, A.; Jirků, Miloslav; Koudela, Břetislav; Gelnar, M.; Modrý, David; Šlapeta, J.

2008-01-01

Roč. 38, 8/9 (2008), s. 913-922 ISSN 0020-7519 R&D Projects: GA ČR GD524/03/H133; GA ČR GA524/05/0992; GA MŠk LC522 Institutional research plan: CEZ:AV0Z60220518 Keywords : Cryptosporidium * host cell invasion * epicellular * parasitophorous sac * ultrastructure Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 3.752, year: 2008

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.

Science.gov (United States)

Quereda, Juan J; Pizarro-Cerdá, Javier; Balestrino, Damien; Bobard, Alexandre; Danckaert, Anne; Aulner, Nathalie; Shorte, Spencer; Enninga, Jost; Cossart, Pascale

2016-01-01

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Polyamines control of cation transport across plant membranes: implications for ion homeostasis and abiotic stress signaling.

Science.gov (United States)

Pottosin, Igor; Shabala, Sergey

2014-01-01

Polyamines are unique polycationic metabolites, controlling a variety of vital functions in plants, including growth and stress responses. Over the last two decades a bulk of data was accumulated providing explicit evidence that polyamines play an essential role in regulating plant membrane transport. The most straightforward example is a blockage of the two major vacuolar cation channels, namely slow (SV) and fast (FV) activating ones, by the micromolar concentrations of polyamines. This effect is direct and fully reversible, with a potency descending in a sequence Spm(4+) > Spd(3+) > Put(2+). On the contrary, effects of polyamines on the plasma membrane (PM) cation and K(+)-selective channels are hardly dependent on polyamine species, display a relatively low affinity, and are likely to be indirect. Polyamines also affect vacuolar and PM H(+) pumps and Ca(2+) pump of the PM. On the other hand, catabolization of polyamines generates H2O2 and other reactive oxygen species (ROS), including hydroxyl radicals. Export of polyamines to the apoplast and their oxidation there by available amine oxidases results in the induction of a novel ion conductance and confers Ca(2+) influx across the PM. This mechanism, initially established for plant responses to pathogen attack (including a hypersensitive response), has been recently shown to mediate plant responses to a variety of abiotic stresses. In this review we summarize the effects of polyamines and their catabolites on cation transport in plants and discuss the implications of these effects for ion homeostasis, signaling, and plant adaptive responses to environment.

Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Jørgensen, M U; Emr, S D; Winther, Jakob R.

1999-01-01

Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand bind...

Knowns and unknowns of plasma membrane protein degradation in plants.

Science.gov (United States)

Liu, Chuanliang; Shen, Wenjin; Yang, Chao; Zeng, Lizhang; Gao, Caiji

2018-07-01

Plasma membrane (PM) not only creates a physical barrier to enclose the intracellular compartments but also mediates the direct communication between plants and the ever-changing environment. A tight control of PM protein homeostasis by selective degradation is thus crucial for proper plant development and plant-environment interactions. Accumulated evidences have shown that a number of plant PM proteins undergo clathrin-dependent or membrane microdomain-associated endocytic routes to vacuole for degradation in a cargo-ubiquitination dependent or independent manner. Besides, several trans-acting determinants involved in the regulation of endocytosis, recycling and multivesicular body-mediated vacuolar sorting have been identified in plants. More interestingly, recent findings have uncovered the participation of selective autophagy in PM protein turnover in plants. Although great progresses have been made to identify the PM proteins that undergo dynamic changes in subcellular localizations and to explore the factors that control the membrane protein trafficking, several questions remain to be answered regarding the molecular mechanisms of PM protein degradation in plants. In this short review article, we briefly summarize recent progress in our understanding of the internalization, sorting and degradation of plant PM proteins. More specifically, we focus on discussing the elusive aspects underlying the pathways of PM protein degradation in plants. Copyright © 2018 Elsevier B.V. All rights reserved.

PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress

Science.gov (United States)

Ding, Jun; Holzwarth, Garrett; Bradford, C. Samuel; Cooley, Ben; Yoshinaga, Allen S.; Patton-Vogt, Jana; Abeliovich, Hagai; Penner, Michael H.; Bakalinsky, Alan T.

2017-01-01

In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity. PMID:26051671

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

Science.gov (United States)

Yeoman, Jeffrey A; Hanssen, Eric; Maier, Alexander G; Klonis, Nectarios; Maco, Bohumil; Baum, Jake; Turnbull, Lynne; Whitchurch, Cynthia B; Dixon, Matthew W A; Tilley, Leann

2011-04-01

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

Hereditary vacuolar internal anal sphincter myopathy causing proctalgia fugax and constipation: a new case contribution.

Science.gov (United States)

de la Portilla, Fernando; Borrero, Juan José; Rafel, Enrique

2005-03-01

Hereditary anal sphincter myopathy is rare. We present a family with one affected member with proctalgia fugax, constipation and internal anal sphincter hypertrophy. Ultrastructural findings show vacuolization of smooth muscle cells without the characteristic polyglucosan inclusion. Further relief of symptoms was obtained using an oral calcium antagonist. Based on clinical presentation, endosonography and morphological findings, we consider our case is a histological variant of the vacuolar myopathy originally described.

TAI vacuolar invertase orthologs: the interspecific variability in tomato plants (Solanum section Lycopersicon).

Science.gov (United States)

Slugina, M A; Shchennikova, A V; Kochieva, E Z

2017-10-01

Understanding the genetic mechanisms underlying carbohydrate metabolism can promote the development of biotechnological advances in fruit plants. The flesh tomato fruit represents an ideal system for examining the role of sucrose cleavage enzymes in fruit development, and wild tomato species differing in storage sugars serve as an excellent research material for this purpose. Plant vacuolar invertase is a key enzyme of sucrose metabolism in the sink organs. In the present study, we identified complete gene sequences encoding the TAI vacuolar invertase in 11 wild and one cultivated tomato accessions of the Solanum section Lycopersicon. The average level of interspecific polymorphism in TAI genes was 8.58%; however, in the green-fruited tomatoes, the TAI genes contained 100 times more SNPs than those in the red-fruited accessions. The TAI proteins demonstrated 8% variability, whereas the red-fruited species had none. A TAI-based phylogenetic tree revealed two main clusters containing self-compatible and self-incompatible species, which concurs with the previous crossability-based division and demonstrates that the TAI genes reflect the evolutionary relationships between the red- and green-fruited tomatoes. Furthermore, we detected differential expression patterns of the TAI genes in the fruits of wild and cultivated tomatoes, which corresponded to sugar composition. The polymorphism analysis of the TAI acid invertases of Solanum section Lycopersicon species will contribute to the understanding of the genetic potential of TAI genes to impact tomato breeding through genetic engineering of the carbohydrate composition in the fruit.

ZIFL1.1 transporter modulates polar auxin transport by stabilizing membrane abundance of multiple PINs in Arabidopsis root tip

Science.gov (United States)

Remy, Estelle; Baster, Pawel; Friml, Jiří; Duque, Paula

2013-01-01

Cell-to-cell directional flow of the phytohormone auxin is primarily established by polar localization of the PIN auxin transporters, a process tightly regulated at multiple levels by auxin itself. We recently reported that, in the context of strong auxin flows, activity of the vacuolar ZIFL1.1 transporter is required for fine-tuning of polar auxin transport rates in the Arabidopsis root. In particular, ZIFL1.1 function protects plasma-membrane stability of the PIN2 carrier in epidermal root tip cells under conditions normally triggering PIN2 degradation. Here, we show that ZIFL1.1 activity at the root tip also promotes PIN1 plasma-membrane abundance in central cylinder cells, thus supporting the notion that ZIFL1.1 acts as a general positive modulator of polar auxin transport in roots. PMID:23857365

Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

Science.gov (United States)

Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

1999-07-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity

DEFF Research Database (Denmark)

Winther, Jakob R.; Stevens, T H; Kielland-Brandt, Morten

1991-01-01

Functions of the carbohydrate side chains of the yeast vacuolar enzyme carboxypeptidase Y (CPY) were investigated by removal, through site-directed mutagenesis, of the sequences which act as signals for N-linked glycosylation. The mutant forms of the enzyme were analysed with respect to activity...

The patatin-like protein from the latex of Hevea brasiliensis (Hev b 7) is not a vacuolar protein

NARCIS (Netherlands)

Jekel, PA; Hofsteenge, J; Beintema, JJ

Upon centrifugation, rubber latex is divided into a layer of rubber particles, the cytosol, and the lutoid-body fraction, which is of vacuolar origin. One of the proteins isolated from the lutoid-body fraction is a protein with a molecular mass of 43 kDa, which has esterase activity on

Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Christopher L Brett

Full Text Available Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pH(v in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pH(v (5.27±0.13 was resistant to acid stress (5.28±0.14 but shifted significantly in response to alkali stress (5.83±0.13. Of 107 mutants that displayed aberrant pH(v under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pH(v dysregulation in a neo1(ts mutant restored viability whereas cholesterol accumulation in human NPC1(-/- fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation.

A systematic analysis of the early transcribed membrane protein family throughout the life cycle of Plasmodium yoelii.

Science.gov (United States)

MacKellar, Drew C; Vaughan, Ashley M; Aly, Ahmed S I; DeLeon, Sasha; Kappe, Stefan H I

2011-11-01

The early transcribed membrane proteins (ETRAMPs) are a family of small, highly charged transmembrane proteins unique to malaria parasites. Some members of the ETRAMP family have been localized to the parasitophorous vacuole membrane that separates the intracellular parasite from the host cell and thus presumably have a role in host-parasite interactions. Although it was previously shown that two ETRAMPs are critical for rodent malaria parasite liver-stage development, the importance of most ETRAMPs during the parasite life cycle remains unknown. Here, we comprehensively identify nine new etramps in the genome of the rodent malaria parasite Plasmodium yoelii, and elucidate their conservation in other malaria parasites. etramp expression profiles are diverse throughout the parasite life cycle as measured by RT-PCR. Epitope tagging of two ETRAMPs demonstrates protein expression in blood and liver stages, and reveals differences in both their timing of expression and their subcellular localization. Gene targeting studies of each of the nine uncharacterized etramps show that two are refractory to deletion and thus likely essential for blood-stage replication. Seven etramps are not essential for any life cycle stage. Systematic characterization of the members of the ETRAMP family reveals the diversity in importance of each family member at the interface between host and parasite throughout the developmental cycle of the malaria parasite. © 2011 Blackwell Publishing Ltd.

Identification of genes affecting vacuole membrane fragmentation in Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Lydie Michaillat

Full Text Available The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

Mutational analysis of the vacuolar sorting signal of procarboxypeptidase Y in yeast shows a low requirement for sequence conservation

DEFF Research Database (Denmark)

van Voorst, F; Kielland-Brandt, Morten; Winther, Jakob R.

1996-01-01

The core of the vacuolar targeting signal of yeast carboxypeptidase Y (CPY) is recognized by the receptor Vps10p and consists of four contiguous amino acid residues, Gln24-Arg-Pro-Leu27, near the amino terminus of the propeptide (Valls, L.A., Winther, J. R., and Stevens, T. H. (1990) J. Cell Biol...

A novel vacuolar membrane H+-ATPase c subunit gene (ThVHAc1) from Tamarix hispida confers tolerance to several abiotic stresses in Saccharomyces cerevisiae.

Science.gov (United States)

Gao, Caiqiu; Wang, Yucheng; Jiang, Bo; Liu, Guifeng; Yu, Lili; Wei, Zhigang; Yang, Chuanping

2011-02-01

Plant vacuolar H(+)-ATPase (V-ATPase) plays an important role in response to different adverse environmental conditions. In the present study, we cloned and characterized a V-ATPase c subunit gene (ThVHAc1) from Tamarix hispida. The deduced ThVHAc1 amino acid sequence lacks a signal peptide and ThVHAc1 is a highly hydrophobic protein with four transmembrane regions. A transient expression assay showed that the ThVHAc1-GFP fusion protein is expressed on onion epidermal endomembrane cells. Real-time RT-PCR demonstrated that ThVHAc1 gene expression was induced by NaCl, NaHCO(3), PEG and CdCl(2) stress in T. hispida roots, stems and leaves. Exogenous application of abscisic acid (ABA) also stimulated ThVHAc1 transcript levels in the absence of stress, suggesting that ThVHAc1 is involved in ABA-dependent stress signaling pathway. Furthermore, the transgenic yeast expressing ThVHAc1 increased salt, drought, ultraviolet (UV), oxidative, heavy metal, cold and high temperature tolerance. Our results suggested that the ThVHAc1 gene from T. hispida serves a stress tolerance role in the species.

Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

Science.gov (United States)

Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

1999-01-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing?

Science.gov (United States)

Wong, Louise H; Levine, Tim P

2016-04-15

Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One type of inter-organelle communication is lipid exchange, which must occur for membrane maintenance and in response to environmental and cellular stimuli. Soluble lipid transfer proteins have been extensively studied, but additional families of transfer proteins have been identified that are anchored into membranes by transmembrane helices so that they cannot diffuse through the cytosol to deliver lipids. If such proteins target membrane contact sites they may be major players in lipid metabolism. The eukaryotic family of so-called Lipid transfer proteins Anchored at Membrane contact sites (LAMs) all contain both a sterol-specific lipid transfer domain in the StARkin superfamily (related to StART/Bet_v1), and one or more transmembrane helices anchoring them in the endoplasmic reticulum (ER), making them interesting subjects for study in relation to sterol metabolism. They target a variety of membrane contact sites, including newly described contacts between organelles that were already known to make contact by other means. Lam1-4p target punctate ER-plasma membrane contacts. Lam5p and Lam6p target multiple contacts including a new category: vacuolar non-NVJ cytoplasmic ER (VancE) contacts. These developments confirm previous observations on tubular lipid-binding proteins (TULIPs) that established the importance of membrane anchored proteins for lipid traffic. However, the question remaining to be solved is the most difficult of all: are LAMs transporters, or alternately are they regulators that affect traffic more indirectly? © 2016 Authors; published by Portland Press Limited.

Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion.

Science.gov (United States)

Martins, Nadini Oliveira; Souza, Renata Torres de; Cordero, Esteban Mauricio; Maldonado, Danielle Cortez; Cortez, Cristian; Marini, Marjorie Mendes; Ferreira, Eden Ramalho; Bayer-Santos, Ethel; Almeida, Igor Correia de; Yoshida, Nobuko; Silveira, José Franco da

2015-11-01

The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions. Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca2+ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion. We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by

Vacuolar-proton-ATPase is involved in the response of cancer cells to ionizing radiation and is a new target for radiosensitization

International Nuclear Information System (INIS)

Hollister, T.C.; Paglin, S.; McMahill, M.S.; Gilles, F.; Yahalom, J.

2001-01-01

Purpose: We have recently described a novel response of human cancer cells to radiation consisting of accumulation of acidic vesicular organelles (AVO) (Cancer Research, 61:439-444, 2001). Acidification of AVO was inhibited by bafilomycin A1 - a specific inhibitor of vacuolar-proton-ATPase. To examine the role of AVO function in the cellular response to radiation, we determined the effect of specific inhibitors of v-H+-ATPase (bafilomycin and concanamycin) on the clonogenic survival of various cancer cell lines. In addition, Northern analysis of mRNA encoding v-H+-ATPase was performed to elucidate possible mechanisms of post-radiation v-H+-ATPase regulation. Materials and Methods: Total RNA was isolated from human cancer cell lines including MCF-7 (breast), LoVo (colon), and LNCaP (prostate) cells 48 hours post-irradiation, and Northern analyses were performed. RNA was separated on a denaturing formaldehyde gel and blotted to a nylon membrane. Probes made from cDNAs to v-H+-ATPase were radioactively labeled, hybridized to the membranes, and exposed to X-ray film for determination of mRNA expression levels. Clonogenic survival assays were performed with MCF-7 and LoVo cell lines. Cells were plated at 30 cells/cm 2 , and irradiated 24 hours post-plating. Bafilomycin or concanamycin were added to culture media in various concentrations at the time of irradiation. The media was changed after a 24-hour incubation, and colonies were stained with crystal violet for counting 7 days post-irradiation. Results: Radiation-induced accumulation of acidic vesicular organelles in MCF-7, LoVo and LNCaP cells was associated with a two-fold increase in the steady-state level of mRNA for subunit c of v-H+-ATPase. Bafilomycin and concanamycin increased clonogenic cell death after irradiation in a dose-dependent manner. At low concentrations (bafilomycin 2nM, concanamycin 2pM) these agents acted as radiosensitizers, without significant toxicity to unirradiated cells. At 2nM, bafilomycin

The assembly of metals chelation by thiols and vacuolar compartmentalization conferred increased tolerance to and accumulation of cadmium and arsenic in transgenic Arabidopsis thaliana

International Nuclear Information System (INIS)

Guo, Jiangbo; Xu, Wenzhong; Ma, Mi

2012-01-01

Highlights: ► Simultaneous transformation of AsPCS1 and ScYCF1 into Arabidopsis thaliana which is sensitive to heavy metals, leads to transgenic plants tolerant to Arsenic and cadmium. ► Dual-gene transgenic Arabidopsis showed higher accumulation of Arsenic and cadmium than single and non-transgenic plants. ► Our results proved that improved thiol peptides synthesis and vacuolar compartmentation in plant dramatically boosted the survival rates of plants when exposed to heavy metals. ► A new strategy for efficient phytoremediation of heavy metals by stacking genes transformation in plants was developed in this article. - Abstract: Transgenic Arabidopsis thaliana were developed to increase tolerance for and accumulation of heavy metals and metalloids by simultaneous overexpression of AsPCS1 and YCF1 (derived from garlic and baker's yeast) based on the fact that chelation of metals and vacuolar compartmentalization are the main strategies for heavy metals/metalloids detoxification and tolerance in plants. Dual-gene transgenic lines had the longest roots and the highest accumulation of Cd and As than single-gene transgenic lines and wildtype. When grown on cadmium or arsenic (arsenite/arsenate), Dual-gene transgenic lines accumulated over 2–10 folds cadmium/arsenite and 2–3 folds arsenate than wild type or plants expressing AsPCS1 or YCF1 alone. Such stacking modified genes involved in chelation of toxic metals and vacuolar compartmentalization represents a highly promising new tool for use in phytoremediation efforts.

Evolution of vacuolar proton pyrophosphatase domains and volutin granules: clues into the early evolutionary origin of the acidocalcisome

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Valerio Alejandro

2011-10-01

Full Text Available Abstract Background Volutin granules appear to be universally distributed and are morphologically and chemically identical to acidocalcisomes, which are electron-dense granular organelles rich in calcium and phosphate, whose functions include storage of phosphorus and various metal ions, metabolism of polyphosphate, maintenance of intracellular pH, osmoregulation and calcium homeostasis. Prokaryotes are thought to differ from eukaryotes in that they lack membrane-bounded organelles. However, it has been demonstrated that as in acidocalcisomes, the calcium and polyphosphate-rich intracellular "volutin granules (polyphosphate bodies" in two bacterial species, Agrobacterium tumefaciens, and Rhodospirillum rubrum, are membrane bound and that the vacuolar proton-translocating pyrophosphatases (V-H+PPases are present in their surrounding membranes. Volutin granules and acidocalcisomes have been found in organisms as diverse as bacteria and humans. Results Here, we show volutin granules also occur in Archaea and are, therefore, present in the three superkingdoms of life (Archaea, Bacteria and Eukarya. Molecular analyses of V-H+PPase pumps, which acidify the acidocalcisome lumen and are diagnostic proteins of the organelle, also reveal the presence of this enzyme in all three superkingdoms suggesting it is ancient and universal. Since V-H+PPase sequences contained limited phylogenetic signal to fully resolve the ancestral nodes of the tree, we investigated the divergence of protein domains in the V-H+PPase molecules. Using Protein family (Pfam database, we found a domain in the protein, PF03030. The domain is shared by 31 species in Eukarya, 231 in Bacteria, and 17 in Archaea. The universal distribution of the V-H+PPase PF03030 domain, which is associated with the V-H+PPase function, suggests the domain and the enzyme were already present in the Last Universal Common Ancestor (LUCA. Conclusion The importance of the V-H+PPase function and the

Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

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Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

2011-02-01

Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

Inhibition of Fusion of Chlamydia trachomatis Inclusions at 32°C Correlates with Restricted Export of IncA

OpenAIRE

Fields, K. A.; Fischer, E.; Hackstadt, T.

2002-01-01

Chlamydia trachomatis is an obligate intracellular bacterium that develops within a parasitophorous vacuole termed an inclusion. The inclusion is nonfusogenic with lysosomes but intercepts lipids from a host cell exocytic pathway. Initiation of chlamydial development is concurrent with modification of the inclusion membrane by a set of C. trachomatis-encoded proteins collectively designated Incs. One of these Incs, IncA, is functionally associated with the homotypic fusion of inclusions. Incl...

Biophysics of malarial parasite exit from infected erythrocytes.

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Chandramohanadas, Rajesh; Park, YongKeun; Lui, Lena; Li, Ang; Quinn, David; Liew, Kingsley; Diez-Silva, Monica; Sung, Yongjin; Dao, Ming; Lim, Chwee Teck; Preiser, Peter Rainer; Suresh, Subra

2011-01-01

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.

Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling

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Jaiswal, Dinesh Kumar; Mishra, Poonam; Subba, Pratigya; Rathi, Divya; Chakraborty, Subhra; Chakraborty, Niranjan

2014-02-01

Dehydration affects almost all the physiological processes including those that result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which in turn elicits a highly conserved signaling, the unfolded protein response (UPR). We investigated the dehydration-responsive membrane-associated proteome of a legume, chickpea, by 2-DE coupled with mass spectrometry. A total of 184 protein spots were significantly altered over a dehydration treatment of 120 h. Among the differentially expressed proteins, a non-canonical SUN domain protein, designated CaSUN1 (Cicer arietinum Sad1/UNC-84), was identified. CaSUN1 localized to the nuclear membrane and ER, besides small vacuolar vesicles. The transcripts were downregulated by both abiotic and biotic stresses, but not by abscisic acid treatment. Overexpression of CaSUN1 conferred stress tolerance in transgenic Arabidopsis. Furthermore, functional complementation of the yeast mutant, slp1, could rescue its growth defects. We propose that the function of CaSUN1 in stress response might be regulated via UPR signaling.

Failure to transmit avian vacuolar myelinopathy to mallard ducks

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Larsen, R.S.; Nutter, F.B.; Augspurger, T.; Rocke, T.E.; Thomas, N.J.; Stoskopf, M.K.

2003-01-01

Avian vacuolar myelinopathy (AVM) is a neurologic disease that has been diagnosed in free-ranging birds in the southeastern United States. Bald eagles (Haliaeetus leuocephalus), American coots (Fulica americana), and mallards (Anas platyrhynchos) have been affected. Previous investigations have not determined the etiology of this disease. In November and December 2002, we attempted to induce AVM in game-farmed mallards through four, 7-day exposure trials. Mallards were housed in six groups of eight, with two of these groups serving as controls. One group was housed with AVM-affected coots; one group was tube fed daily with water from the lake where affected coots were captured; one group was tube fed daily with aquatic vegetation (Hydrilla verticillata) from the same lake; and another group was tube fed daily with sediment from the lake. No ducks exhibited clinical neurologic abnormalities consistent with AVM and no evidence of AVM was present at histopathologic examination of brain tissue. Although limitations in sample size, quantity of individual doses, frequency of dose administration, duration of exposure, and timing of these trials restrict the interpretation of the findings, AVM was not readily transmitted by direct contact, water, hydrilla, or sediment in this investigation.

TBK1 protects vacuolar integrity during intracellular bacterial infection.

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Andrea L Radtke

2007-03-01

Full Text Available TANK-binding kinase-1 (TBK1 is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella, more extensive bacterial proliferation was observed in tbk1(-/- than tbk1(+/+ cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1(-/-cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity.

TBK1 Protects Vacuolar Integrity during Intracellular Bacterial Infection

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Radtke, Andrea L; Delbridge, Laura M; Balachandran, Siddharth; Barber, Glen N; O'Riordan, Mary X. D

2007-01-01

TANK-binding kinase-1 (TBK1) is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella), more extensive bacterial proliferation was observed in tbk1−/− than tbk1+/+ cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1−/−cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity. PMID:17335348

The assembly of metals chelation by thiols and vacuolar compartmentalization conferred increased tolerance to and accumulation of cadmium and arsenic in transgenic Arabidopsis thaliana

Energy Technology Data Exchange (ETDEWEB)

Guo, Jiangbo [Key Laboratory of Plant Resources, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093 (China); Inner Mongolia Key Laboratory of Biomass-Energy Conversion, The Institute of Bioengineering and Technology, Inner Mongolia University of Science and Technology, Baotou 040100 (China); Xu, Wenzhong [Key Laboratory of Plant Resources, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093 (China); Ma, Mi, E-mail: mami@ibcas.ac.cn [Key Laboratory of Plant Resources, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093 (China)

2012-01-15

Highlights: Black-Right-Pointing-Pointer Simultaneous transformation of AsPCS1 and ScYCF1 into Arabidopsis thaliana which is sensitive to heavy metals, leads to transgenic plants tolerant to Arsenic and cadmium. Black-Right-Pointing-Pointer Dual-gene transgenic Arabidopsis showed higher accumulation of Arsenic and cadmium than single and non-transgenic plants. Black-Right-Pointing-Pointer Our results proved that improved thiol peptides synthesis and vacuolar compartmentation in plant dramatically boosted the survival rates of plants when exposed to heavy metals. Black-Right-Pointing-Pointer A new strategy for efficient phytoremediation of heavy metals by stacking genes transformation in plants was developed in this article. - Abstract: Transgenic Arabidopsis thaliana were developed to increase tolerance for and accumulation of heavy metals and metalloids by simultaneous overexpression of AsPCS1 and YCF1 (derived from garlic and baker's yeast) based on the fact that chelation of metals and vacuolar compartmentalization are the main strategies for heavy metals/metalloids detoxification and tolerance in plants. Dual-gene transgenic lines had the longest roots and the highest accumulation of Cd and As than single-gene transgenic lines and wildtype. When grown on cadmium or arsenic (arsenite/arsenate), Dual-gene transgenic lines accumulated over 2-10 folds cadmium/arsenite and 2-3 folds arsenate than wild type or plants expressing AsPCS1 or YCF1 alone. Such stacking modified genes involved in chelation of toxic metals and vacuolar compartmentalization represents a highly promising new tool for use in phytoremediation efforts.

Sequence Classification: 889075 [

Lifescience Database Archive (English)

Full Text Available localizes to vacuolar membranes and punctate structures attached to the vacuole; Atg22p || http://www.ncbi.nlm.nih.gov/protein/6319811 ... ...he breakdown of autophagic vesicles in the vacuole during autophagy, putative integral membrane protein that

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue.

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Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery.

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Poëa-Guyon, Sandrine; Ammar, Mohamed Raafet; Erard, Marie; Amar, Muriel; Moreau, Alexandre W; Fossier, Philippe; Gleize, Vincent; Vitale, Nicolas; Morel, Nicolas

2013-10-28

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells.

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Avila, Emily L; Brown, Michelle; Pan, Songqin; Desikan, Radhika; Neill, Steven J; Girke, Thomas; Surpin, Marci; Raikhel, Natasha V

2008-01-01

Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting approximately 1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 degrees C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase Dgamma, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress.

Cadmium induces changes in sucrose partitioning, invertase activities, and membrane functionality in roots of Rangpur lime (Citrus limonia L. Osbeck).

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Podazza, G; Rosa, M; González, J A; Hilal, M; Prado, F E

2006-09-01

Cadmium (Cd) uptake effects on sucrose content, invertase activities, and plasma membrane functionality were investigated in Rangpur lime roots ( CITRUS LIMONIA L. Osbeck). Cadmium accumulation was significant in roots but not in shoots and leaves. Cadmium produced significant reduction in roots DW and increment in WC. Leaves and shoots did not show significant differences on both parameters. Sucrose content was higher in control roots than in Cd-exposed ones. Apoplastic sucrose content was much higher in Cd-exposed roots than in control ones. Cd-exposed roots showed a significant decrease in both cell wall-bound and cytoplasmic (neutral) invertase activities; while the vacuolar isoform did not show any change. Alterations in lipid composition and membrane fluidity of Cd-exposed roots were also observed. In Cd-exposed roots phospholipid and glycolipid contents decreased about 50 %, while sterols content was reduced about 22 %. Proton extrusion was inhibited by Cd. Lipid peroxidation and proton extrusion inhibition were also detected by histochemical analysis. This work's findings demonstrate that Cd affects sucrose partitioning and invertase activities in apoplastic and symplastic regions in Rangpur lime roots as well as the plasma membrane functionality and H (+)-ATPase activity.

Vacuolar Protein Sorting Genes in Parkinson's Disease: A Re-appraisal of Mutations Detection Rate and Neurobiology of Disease.

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Gambardella, Stefano; Biagioni, Francesca; Ferese, Rosangela; Busceti, Carla L; Frati, Alessandro; Novelli, Giuseppe; Ruggieri, Stefano; Fornai, Francesco

2016-01-01

Mammalian retromers play a critical role in protein trans-membrane sorting from endosome to the trans-Golgi network (TGN). Recently, retromer alterations have been related to the onset of Parkinson's Disease (PD) since the variant p.Asp620Asn in VPS35 (Vacuolar Protein Sorting 35) was identified as a cause of late onset PD. This variant causes a primary defect in endosomal trafficking and retromers formation. Other mutations in VPS genes have been reported in both sporadic and familial PD. These mutations are less defined. Understanding the specific prevalence of all VPS gene mutations is key to understand the relevance of retromers impairment in the onset of PD. A number of PD-related mutations despite affecting different biochemical systems (autophagy, mitophagy, proteasome, endosomes, protein folding), all converge in producing an impairment in cell clearance. This may explain how genetic predispositions to PD may derive from slightly deleterious VPS mutations when combined with environmental agents overwhelming the clearance of the cell. This manuscript reviews genetic data produced in the last 5 years to re-define the actual prevalence of VPS gene mutations in the onset of PD. The prevalence of p.Asp620Asn mutation in VPS35 is 0.286 of familial PD. This increases up to 0.548 when considering mutations affecting all VPS genes. This configures mutations in VPS genes as the second most frequent autosomal dominant PD genotype. This high prevalence, joined with increased awareness of the role played by retromers in the neurobiology of PD, suggests environmentally-induced VPS alterations as crucial in the genesis of PD.

Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

Science.gov (United States)

Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

2014-01-01

The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

Arabidopsis senescence-associated protein DMP1 is involved in membrane remodeling of the ER and tonoplast

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Kasaras Alexis

2012-04-01

Full Text Available Abstract Background Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant-specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression. Results Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER and the vacuole. Induction of spherical structures (“bulbs”, changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed “boluses” in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis. Conclusions Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1

Apoptosis Gene Hunting Using Retroviral Expression Cloning: Identification of Vacuolar ATPase Subunit E

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Claire L. Anderson

2003-01-01

Full Text Available Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E.

Regulation of vacuolar H+-ATPase in microglia by RANKL

International Nuclear Information System (INIS)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F.; Holliday, L. Shannon

2009-01-01

Vacuolar H + -ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor κB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor κB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

Energy Technology Data Exchange (ETDEWEB)

Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.; Marceau, François, E-mail: francois.marceau@crchul.ulaval.ca

2013-07-15

Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K{sub M} 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V{sub max}. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V-ATPase-mediated ion

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

International Nuclear Information System (INIS)

Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.; Marceau, François

2013-01-01

Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K M 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V max . PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V-ATPase-mediated ion trapping

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

Science.gov (United States)

Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

2008-01-01

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

The "Arabidopsis cax3" mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H(+)-ATPase activity

Science.gov (United States)

Perturbing CAX1, an "Arabidopsis" vacuolar H(+)/Ca(2+) antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among "cax1" and "ca...

Vacuolar Protein Sorting Genes in Parkinson's Disease: A Re-appraisal of Mutations Detection Rate and Neurobiology of Disease

Science.gov (United States)

Gambardella, Stefano; Biagioni, Francesca; Ferese, Rosangela; Busceti, Carla L.; Frati, Alessandro; Novelli, Giuseppe; Ruggieri, Stefano; Fornai, Francesco

2016-01-01

Mammalian retromers play a critical role in protein trans-membrane sorting from endosome to the trans-Golgi network (TGN). Recently, retromer alterations have been related to the onset of Parkinson's Disease (PD) since the variant p.Asp620Asn in VPS35 (Vacuolar Protein Sorting 35) was identified as a cause of late onset PD. This variant causes a primary defect in endosomal trafficking and retromers formation. Other mutations in VPS genes have been reported in both sporadic and familial PD. These mutations are less defined. Understanding the specific prevalence of all VPS gene mutations is key to understand the relevance of retromers impairment in the onset of PD. A number of PD-related mutations despite affecting different biochemical systems (autophagy, mitophagy, proteasome, endosomes, protein folding), all converge in producing an impairment in cell clearance. This may explain how genetic predispositions to PD may derive from slightly deleterious VPS mutations when combined with environmental agents overwhelming the clearance of the cell. This manuscript reviews genetic data produced in the last 5 years to re-define the actual prevalence of VPS gene mutations in the onset of PD. The prevalence of p.Asp620Asn mutation in VPS35 is 0.286 of familial PD. This increases up to 0.548 when considering mutations affecting all VPS genes. This configures mutations in VPS genes as the second most frequent autosomal dominant PD genotype. This high prevalence, joined with increased awareness of the role played by retromers in the neurobiology of PD, suggests environmentally-induced VPS alterations as crucial in the genesis of PD. PMID:27932943

The Presynaptic v-ATPase Reversibly Disassembles and Thereby Modulates Exocytosis but Is Not Part of the Fusion Machinery

Directory of Open Access Journals (Sweden)

Anna Bodzęta

2017-08-01

Full Text Available Vacuolar H+-ATPase (v-ATPase is a multi-subunit complex comprising two domains: the cytosolic V1 domain catalyzing ATP hydrolysis and the membranous V0 sector translocating protons across membranes. In addition to proton pumping, a direct function of the V0 proteolipid ring in membrane fusion has been proposed for yeast vacuolar fusion and synaptic vesicle exocytosis in Drosophila. Here, we show in cultured hippocampal neurons that in recycling synaptic vesicles, v-ATPases are only transiently assembled in a pH-dependent fashion during the tightly coupled cycle of exo-endocytosis. Upon locking v-ATPase in an assembled state by saliphenylhalamide, we observed use- and time-dependent release depression for stimuli exceeding release of primed vesicles but no abrogation of exocytosis. Thus, the membranous V0 sector is not part of the exocytotic fusion machinery. Instead, v-ATPase modulates release upstream of docking to favor fusion of fully filled synaptic vesicles.

Mielite transversa como manifestação clínica inicial de linfoma não Hodgkin disseminado e mielopatia vacuolar associada ao HIV: relato de caso Transverse myelitis as initial symptom of disseminated non-Hodgkin lymphoma and HIV-associated vacuolar myelopathy: case report

Directory of Open Access Journals (Sweden)

Leandro P. de Moura

1996-06-01

Full Text Available Linfomas não Hodgkin de alto grau são comumente relatados em pacientes com a síndrome da imunodeficiência adquirida (AIDS. Comprometendo com grande freqüência o sistema nervoso central, particularmente as leptomeninges e os hemisférios cerebrais. O acometimento epidural é pouco freqüente, variando de 3,5% a 8,3% de acordo com os registros da literatura. Os autores relatam o caso de um paciente de 27 anos de idade com AIDS, cuja manifestação clínica inicial da doença linfomatosa disseminada foi a mielite transversa associada à mielopatia vacuolar. Destaca-se a importância do diagnóstico diferencial precoce das mielopatias na AIDS, em virtude da alta malignidade da neoplasia e da evolução extremamente rápida nesses pacientes.Non-Hodgkin lymphoma is frequently seen in AIDS patients usually affecting the central nervous system (CNS, especially the leptomeninges and the cerebral hemispheres. The epidural involvement is rarely described, ranging from 3.5% to 8.3% among the CNS sites. The authors present a case of disseminated non Hodgkin lymphoma associated to vacuolar myelopathy in a 27 years-old male patient with AIDS emphasizing the importance of this differential diagnosis in the myelopathies of AIDS.

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

Science.gov (United States)

Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

1995-10-01

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.

Vacuolar-type H⁺-ATPase and Na⁺, K⁺-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water

DEFF Research Database (Denmark)

Seidelin, Michel; Brauner, Colin J; Jensen, Frank Bo

2001-01-01

Changes in branchial vacuolar-type H+-ATPase B-subunit mRNA and Na+, K+-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days...... Na+, K+-ATPase activity was generally unaffected by the experimental treatments. We suggest that the reduced expression of branchial vacuolar-type H(+)-ATPase B-subunit mRNA observed during internal hypercapnic acidosis may lead to reduction of functional V-type H+-ATPase abundance as a compensatory...

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

Science.gov (United States)

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

2011-10-01

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

Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

Directory of Open Access Journals (Sweden)

Narendranath Reddy Chintagari

2010-02-01

Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

ORF Alignment: NC_001144 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... unconjugated bilirubin and in heavy metal detoxification ... via glutathione conjugates, al...mbrane transporter of MRP/CFTR family, found in ... vacuolar membrane, involved in the transport of ...

Differential activity of Plasma and Vacuolar Membrane Transporters contributes to Genotypic Differences in Salinity Tolerance in a Halophyte Species, Chenopodium quinoa

DEFF Research Database (Denmark)

Bonales-Alatorre, Edgar; Pottosin, Igor; Shabala, Lana

2013-01-01

quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa....... These include: (i) a higher rate of Na+ exclusion from leaf mesophyll; (ii) maintenance of low cytosolic Na+ levels; (iii) better K+ retention in the leaf mesophyll; (iv) a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v) the ability to reduce......Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd.), a facultative C3 halophyte species, can efficiently control the activity of slow...

Vacuolar Protein Sorting genes in Parkinson’s Disease: a re-appraisal of mutations detection rate and neurobiology of disease

Directory of Open Access Journals (Sweden)

Stefano Gambardella

2016-11-01

Full Text Available Mammalian retromers play a critical role in protein trans-membrane sorting from endosome to the trans-Golgi network (TGN. Recently, retromers have been linked to Parkinson's Disease (PD since the identification of the variant p.Asp620Asn in VPS35 (Vacuolar Protein Sorting 35 as a cause of late onset PD. This variant causes a primary defect in endosomal trafficking and retromers formation, which represent critical steps in the molecular mechanisms of disease. Other slightly penetrant and mildly deleterious mutations in VPS genes have been reported in both sporadic and familial PD. Therefore, understanding the actual prevalence of the whole range of VPS gene mutations is key to understand the relevance of retromers impairment in PD. This scenario indicates a plethora of mutations occurring in different pathways (autophagy, mitophagy, proteasome, endosomes, protein misfolding all converging to cell clearing systems. This may explain how genetic predispositions to PD may derive from slightly deleterious mutations when combining with heterogeneous environmental factors. This manuscript is a re-appraisal of genetic data produced in the last five years redefining the prevalence of VPS mutations in PD. The prevalence of p.Asp620Asn in VPS35 is 0.286 of familial PD. This data increases up to 0.548 considering mutations affecting all VPS genes, thus representing the second most frequent autosomal dominant PD genotype. This high prevalence, joined with increased awareness of the key role of retromers alterations in PD, strongly candidate environmentally-induced VPS alterations as key molecular mechanisms in the genesis of PD. rations as key molecular mechanisms in the genesis of PD.

The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion

DEFF Research Database (Denmark)

Takeda, Kozue; Cabrera, Margarita; Rohde, Jan

2008-01-01

At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate...

Sequence Classification: 892097 [

Lifescience Database Archive (English)

Full Text Available into the multivesicular body pathway to the lysosomal/vacuolar lumen; cytoplasmic protein recruited to endosomal membranes; Vps20p || http://www.ncbi.nlm.nih.gov/protein/6323723 ...

Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells.

Science.gov (United States)

Thayer, S S; Huffaker, R C

1984-05-01

The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

Regulation of transport processes across the tonoplast

Science.gov (United States)

Neuhaus, H. Ekkehard; Trentmann, Oliver

2014-01-01

In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g., due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation. PMID:25309559

Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

Science.gov (United States)

The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1– expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and p...

Endocytosis regulates membrane localization and function of the fusogen EFF-1.

Science.gov (United States)

Smurova, Ksenia; Podbilewicz, Benjamin

2017-07-03

Cell fusion is essential for sexual reproduction and formation of muscles, bones, and placenta. Two families of cell fusion proteins (Syncytins and FFs) have been identified in eukaryotes. Syncytins have been shown to form the giant syncytial trophoblasts in the placenta. The FFs are essential to fuse cells in the skin, reproductive, excretory, digestive and nervous systems in nematodes. EFF-1 (Epithelial Fusion Failure 1), a member of the FF family, is a type I membrane glycoprotein that is essential for most cell fusions in C. elegans. The crystal structure of EFF-1 ectodomain reveals striking structural similarity to class II fusion glycoproteins from enveloped viruses (e.g. dengue and rubella) that mediate virus to cell fusion. We found EFF-1 to be present on the plasma membrane and in RAB-5-positive early endosomes, with EFF-1 recycling between these 2 cell compartments. Only when EFF-1 proteins transiently arrive to the surfaces of 2 adjacent cells do they dynamically interact in trans and mediate membrane fusion. EFF-1 is continuously internalized by receptor-mediated endocytosis via the activity of 2 small GTPases: RAB-5 and Dynamin. Here we propose a model that explains how EFF-1 endocytosis together with interactions in trans can control cell-cell fusion. Kontani et al. showed that vacuolar ATPase (vATPase) mutations result in EFF-1-dependent hyperfusion. 1 We propose that vATPase is required for normal degradation of EFF-1. Failure to degrade EFF-1 results in delayed hyperfusion and mislocalization to organelles that appear to be recycling endosomes. EFF-1 is also required to fuse neurons as part of the repair mechanism following injury and to prune dendrites. We speculate that EFF-1 may regulate neuronal tree like structures via endocytosis. Thus, endocytosis of cell-cell fusion proteins functions to prevent merging of cells and to sculpt organs and neurons.

Effects of X-ray irradiation combined with hyperthermia on human bone marrow cells

International Nuclear Information System (INIS)

Xie Huaijiang; Niu Rongjiu; Liu Xiaodong; Liu Huanqin

1996-01-01

The authors report on the effects of X-ray irradiation combined with hyperthermia on human bone marrow cells (BMC) in vitro. Observation was made on the morphology of treated cells under optic microscope and ultrastructural changes under electron microscope. The change was not obvious at first after treatment i,e, only the vacuolar degeneration was observed in a few cells under the EM. The survival of BMC alone after irradiation decreased with increase of the irradiation dose. The morphological changes included vacuolar degeneration of cells, swelling of mitochondria, and disintegration of nuclear membranes. The survival rate of BMC after irradiation combined with hyperthermia was significantly lower than that after treatment by either of them alone (P<0.01). The morphological changes were as follows: the cell structure was destroyed, the cell support system and cell organelles were destroyed, the cell membrane and nuclear membranes were destroyed, and the cell plasma and nuclear sap overflowed

Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

Science.gov (United States)

Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

1996-01-01

Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

Science.gov (United States)

Luna-Tapia, Arturo; Peters, Brian M; Eberle, Karen E; Kerns, Morgan E; Foster, Timothy P; Marrero, Luis; Noverr, Mairi C; Fidel, Paul L; Palmer, Glen E

2015-10-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Vps1 in the late endosome-to-vacuole traffic

Indian Academy of Sciences (India)

2013-01-13

Jan 13, 2013 ... the plasma membrane lipids and follows the endocytic inter- nalization pathways ... plasm and found also as punctuate structures (figure 1A). Our explanation ..... 1992 Morphological classification of the yeast vacuolar protein.

Yeast Interacting Proteins Database: YKL002W, YLR423C [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthes... into lumenal vesicles of multivesicular bodies, and for delivery of newly synthesized vacuolar enzymes to t

Yeast Interacting Proteins Database: YKL002W, YDL165W [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthes...ins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthesized vacuolar enzymes t

Yeast Interacting Proteins Database: YER081W, YDR105C [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available YDR105C TMS1 Vacuolar membrane protein of unknown function that is conserved in mammals; predicted to contai...tion that is conserved in mammals; predicted to contain eleven transmembrane heli

Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

Energy Technology Data Exchange (ETDEWEB)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: sholliday@dental.ufl.edu [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)

2009-11-06

Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

Suppressors for Human Epidermal Growth Factor Receptor 2/4 (HER2/4): A New Family of Anti-Toxoplasmic Agents in ARPE-19 Cells.

Science.gov (United States)

Kim, Yeong Hoon; Bhatt, Lokraj; Ahn, Hye-Jin; Yang, Zhaoshou; Lee, Won-Kyu; Nam, Ho-Woo

2017-10-01

The effects of tyrosine kinase inhibitors (TKIs) were evaluated on growth inhibition of intracellular Toxoplasma gondii in host ARPE-19 cells. The number of tachyzoites per parasitophorous vacuolar membrane (PVM) was counted after treatment with TKIs. T. gondii protein expression was assessed by western blot. Immunofluorescence assay was performed using Programmed Cell Death 4 (PDCD4) and T. gondii GRA3 antibodies. The TKIs were divided into 3 groups; non-epidermal growth factor receptor (non-EGFR), anti-human EGFR 2 (anti-HER2), and anti-HER2/4 TKIs, respectively. Group I TKIs (nintedanib, AZD9291, and sunitinib) were unable to inhibit proliferation without destroying host cells. Group II TKIs (lapatinib, gefitinib, erlotinib, and AG1478) inhibited proliferation up to 98% equivalent to control pyrimethamine (5 μM) at 20 μM and higher, without affecting host cells. Group III TKIs (neratinib, dacomitinib, afatinib, and pelitinib) inhibited proliferation up to 98% equivalent to pyrimethamine at 1-5 μM, but host cells were destroyed at 10-20 μM. In Group I, TgHSP90 and SAG1 inhibitions were weak, and GRA3 expression was moderately inhibited. In Group II, TgHSP90 and SAG1 expressions seemed to be slightly enhanced, while GRA3 showed none to mild inhibition; however, AG1478 inhibited all proteins moderately. Protein expression was blocked in Group III, comparable to pyrimethamine. PDCD4 and GRA3 were well localized inside the nuclei in Group I, mildly disrupted in Group II, and were completely disrupted in Group III. This study suggests the possibility of a vital T. gondii TK having potential HER2/4 properties, thus anti-HER2/4 TKIs may inhibit intracellular parasite proliferation with minimal adverse effects on host cells.

The rhoptry proteome of Eimeria tenella sporozoites

KAUST Repository

Oakes, Richard D.; Kurian, Dominic; Bromley, Elizabeth V.; Ward, Chris; Lal, Kalpana; Blake, Damer P.; Reid, Adam James; Pain, Arnab; Sinden, Robert E.; Wastling, Jonathan M.; Tomley, F. M. M Fiona

2013-01-01

Proteins derived from the rhoptry secretory organelles are crucial for the invasion and survival of apicomplexan parasites within host cells. The rhoptries are club-shaped organelles that contain two distinct subpopulations of proteins that localise to separate compartments of the organelle. Proteins from the neck region (rhoptry neck proteins, RON) are secreted early in invasion and a subset of these is critical for the formation and function of the moving junction between parasite and host membranes. Proteins from the bulb compartment (rhoptry protein, ROP) are released later, into the nascent parasitophorous vacuole where they have a role in modifying the vacuolar environment, and into the host cell where they act as key determinants of virulence through their ability to interact with host cell signalling pathways, causing an array of downstream effects. In this paper we present the results of an extensive proteomics analysis of the rhoptry organelles from the coccidian parasite, Eimeria tenella, which is a highly pathogenic parasite of the domestic chicken causing severe caecal coccidiosis. Several different classes of rhoptry protein have been identified. First are the RON proteins that have varying degrees of similarity to proteins of Toxoplasma gondii and Neospora caninum. For some RON families, E. tenella expresses more than one gene product and many of the individual RON proteins are differentially expressed between the sporozoite and merozoite developmental stages. The E. tenella sporozoite rhoptry expresses only a limited repertoire of proteins with homology to known ROP proteins from other coccidia, including just two secreted ROP kinases, both of which appear to be equipped for catalytic activity. Finally, a large number of hitherto undescribed proteins that map to the sporozoite rhoptry are identified, many of which have orthologous proteins encoded within the genomes of T. gondii and N. caninum. © 2012 .

The rhoptry proteome of Eimeria tenella sporozoites

KAUST Repository

Oakes, Richard D.

2013-02-01

Proteins derived from the rhoptry secretory organelles are crucial for the invasion and survival of apicomplexan parasites within host cells. The rhoptries are club-shaped organelles that contain two distinct subpopulations of proteins that localise to separate compartments of the organelle. Proteins from the neck region (rhoptry neck proteins, RON) are secreted early in invasion and a subset of these is critical for the formation and function of the moving junction between parasite and host membranes. Proteins from the bulb compartment (rhoptry protein, ROP) are released later, into the nascent parasitophorous vacuole where they have a role in modifying the vacuolar environment, and into the host cell where they act as key determinants of virulence through their ability to interact with host cell signalling pathways, causing an array of downstream effects. In this paper we present the results of an extensive proteomics analysis of the rhoptry organelles from the coccidian parasite, Eimeria tenella, which is a highly pathogenic parasite of the domestic chicken causing severe caecal coccidiosis. Several different classes of rhoptry protein have been identified. First are the RON proteins that have varying degrees of similarity to proteins of Toxoplasma gondii and Neospora caninum. For some RON families, E. tenella expresses more than one gene product and many of the individual RON proteins are differentially expressed between the sporozoite and merozoite developmental stages. The E. tenella sporozoite rhoptry expresses only a limited repertoire of proteins with homology to known ROP proteins from other coccidia, including just two secreted ROP kinases, both of which appear to be equipped for catalytic activity. Finally, a large number of hitherto undescribed proteins that map to the sporozoite rhoptry are identified, many of which have orthologous proteins encoded within the genomes of T. gondii and N. caninum. © 2012 .

Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae.

OpenAIRE

Srivastava, A; Woolford, C A; Jones, E W

2000-01-01

Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional ...

Impact of AtNHX1, a vacuolar Na+/H+ antiporter, upon gene expression during short- and long-term salt stress in Arabidopsis thaliana

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Blumwald Eduardo

2007-04-01

Full Text Available Abstract Background AtNHX1, the most abundant vacuolar Na+/H+ antiporter in Arabidopsis thaliana, mediates the transport of Na+ and K+ into the vacuole, influencing plant development and contributing to salt tolerance. In this report, microarray expression profiles of wild type plants, a T-DNA insertion knockout mutant of AtNHX1 (nhx1, and a 'rescued' line (NHX1::nhx1 were exposed to both short (12 h and 48 h and long (one and two weeks durations of a non-lethal salt stress to identify key gene transcripts associated with the salt response that are influenced by AtNHX1. Results 147 transcripts showed both salt responsiveness and a significant influence of AtNHX1. Fifty-seven of these genes showed an influence of the antiporter across all salt treatments, while the remaining genes were influenced as a result of a particular duration of salt stress. Most (69% of the genes were up-regulated in the absence of AtNHX1, with the exception of transcripts encoding proteins involved with metabolic and energy processes that were mostly down-regulated. Conclusion While part of the AtNHX1-influenced transcripts were unclassified, other transcripts with known or putative roles showed the importance of AtNHX1 to key cellular processes that were not necessarily limited to the salt stress response; namely calcium signaling, sulfur metabolism, cell structure and cell growth, as well as vesicular trafficking and protein processing. Only a small number of other salt-responsive membrane transporter transcripts appeared significantly influenced by AtNHX1.

[Effect of adaptogenic preparations on Na+/H+-antiporter function in plasma membrane of corn root cells under salinity conditions].

Science.gov (United States)

Kovalenko, N O; Bilyk, Zh I; Palladina, T O

2014-01-01

Salinity is a hard stress factor for plant organisms which negative effect is caused chiefly by sodium toxic for plants. Plant cells try to remove Na+ from their cytoplasm outside and to vacuolar space by secondary active Na+/H+-antiporters. Their functions can be intensified by gene engineering methods however we try do it with the help of non-toxic bioactive preparations. A comparison of their effect on the plasma membrane of Na+/H+-antiporters was carried out on corn seedling roots of Zea mays L. exposed at 0.1 M NaCl. Before we have established that Methyure used by seed pretreating possesses a high salt protective ability as against Ivine. It was found that without NaCl exposition Na+/H+-antiporter activity in root plasma membrane was nearly unnoticeable but increased slightly with seedling age. Methyure and Ivine did not influence its activity in control root seedling. One day 0.1 M NaCl exposition evoked a considerable increasing of Na+/H+-antiporter activity and its gene expression but these effects disappeared at 10 day NaCl exposition. Methyure use reinforced Na+/H+-antiporter activity and prolonged it at NaCl exposition without effect on its gene expression whereas Ivine effects on these indexes were insignificant. Obtained results showed that the salt protective capability of Methyure is connected with plasma membrane Na+/H+-antiporter activation which is realized on molecular level.

Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

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Coreen Johnson

Full Text Available Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

Stress regulated members of the plant organic cation transporter family are localized to the vacuolar membrane

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Koch Wolfgang

2008-07-01

Full Text Available Abstract Background In Arabidopsis six genes group into the gene family of the organic cation transporters (OCTs. In animals the members of the OCT-family are mostly characterized as polyspecific transporters involved in the homeostasis of solutes, the transport of monoamine neurotransmitters and the transport of choline and carnitine. In plants little is known about function, localisation and regulation of this gene family. Only one protein has been characterized as a carnitine transporter at the plasma membrane so far. Findings We localized the five uncharacterized members of the Arabidopsis OCT family, designated OCT2-OCT6, via GFP fusions and protoplast transformation to the tonoplast. Expression analysis with RNA Gel Blots showed a distinct, organ-specific expression pattern of the individual genes. With reporter gene fusion of four members we analyzed the tissue specific distribution of OCT2, 3, 4, and 6. In experiments with salt, drought and cold stress, we could show that AtOCT4, 5 and 6 are up-regulated during drought stress, AtOCT3 and 5 during cold stress and AtOCT 5 and 6 during salt stress treatments. Conclusion Localisation of the proteins at the tonoplast and regulation of the gene expression under stress conditions suggests a specific role for the transporters in plant adaptation to environmental stress.

Receptor-independent, vacuolar ATPase-mediated cellular uptake of histamine receptor-1 ligands: Possible origin of pharmacological distortions and side effects

International Nuclear Information System (INIS)

Morissette, Guillaume; Lodge, Robert; Bouthillier, Johanne; Marceau, Francois

2008-01-01

The aims of this study were to investigate whether several histamine receptor agonists and antagonists are subjected to receptor-independent ion trapping into acidic organelles, and whether this sequestration influences their pharmacological or toxicological properties. Vacuolar (V)-ATPase-dependent intracellular sequestration of agonists was recognized as morphological alterations (large fluid-filled vacuoles for betahistine and 1-methylhistamine, granular uptake for fluorescent BODIPY FL histamine) prevented by the specific V-ATPase inhibitor bafilomycin A1 in rabbit vascular smooth muscle cells. Lipophilicity was the major determinant of these cellular effects (order of potency: BODIPY FL histamine > betahistine > 1-methylhistamine > histamine) that occurred at high concentrations. This ranking was dissociable from the potency order for H 1 receptor-mediated contraction of the rabbit aorta, a response uninfluenced by bafilomycin. Antihistamines are inherently more lipophilic and caused vacuolization of a proportion of cells at 5-500 μM. Agonist or antagonist-induced vacuoles were of macroautophagic nature (labeled with GFP-conjugated LC3, Rab7 and CD63; detection of LC3 II). Further, the 2 most lipophilic antihistamines tested, astemizole and terfenadine, were potentiated by V-ATPase blockade in the aortic contractility assay (13- and 3.6-fold more potent, respectively, pA 2 scale), suggesting that V-ATPase-mediated cation trapping sequesters these antagonists from the vicinity of H 1 receptors in the therapeutic concentration range. This potentiation did not apply to less lipophilic antagonists (pyrilamine, diphenhydramine). While some agonists and all tested antagonists of the histamine H 1 receptors induce the V-ATPase-dependent vacuolar and autophagic cytopathology, sequestration affects the pharmacology of only the most lipophilic antagonists, the ones prone to off-target arrhythmogenic side effects

Differential Activity of Plasma and Vacuolar Membrane Transporters Contributes to Genotypic Differences in Salinity Tolerance in a Halophyte Species, Chenopodium quinoa

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Edgar Bonales-Alatorre

2013-04-01

Full Text Available Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd., a facultative C3 halophyte species, can efficiently control the activity of slow (SV and fast (FV tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013 Plant Physiology. This work extends these finding by comparing the properties of tonoplast FV and SV channels in two quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa. These include: (i a higher rate of Na+ exclusion from leaf mesophyll; (ii maintenance of low cytosolic Na+ levels; (iii better K+ retention in the leaf mesophyll; (iv a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v the ability to reduce the activity of SV and FV channels under saline conditions. These mechanisms appear to be highly orchestrated, thus enabling the remarkable overall salinity tolerance of quinoa species.

Differential activity of plasma and vacuolar membrane transporters contributes to genotypic differences in salinity tolerance in a Halophyte Species, Chenopodium quinoa.

Science.gov (United States)

Bonales-Alatorre, Edgar; Pottosin, Igor; Shabala, Lana; Chen, Zhong-Hua; Zeng, Fanrong; Jacobsen, Sven-Erik; Shabala, Sergey

2013-04-29

Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd.), a facultative C3 halophyte species, can efficiently control the activity of slow (SV) and fast (FV) tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013) Plant Physiology). This work extends these finding by comparing the properties of tonoplast FV and SV channels in two quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa. These include: (i) a higher rate of Na+ exclusion from leaf mesophyll; (ii) maintenance of low cytosolic Na+ levels; (iii) better K+ retention in the leaf mesophyll; (iv) a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v) the ability to reduce the activity of SV and FV channels under saline conditions. These mechanisms appear to be highly orchestrated, thus enabling the remarkable overall salinity tolerance of quinoa species.

Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

Science.gov (United States)

Novikova, Galina V; Tournaire-Roux, Colette; Sinkevich, Irina A; Lityagina, Snejana V; Maurel, Christophe; Obroucheva, Natalie

2014-09-01

A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Morphological classification of plant cell deaths.

Science.gov (United States)

van Doorn, W G; Beers, E P; Dangl, J L; Franklin-Tong, V E; Gallois, P; Hara-Nishimura, I; Jones, A M; Kawai-Yamada, M; Lam, E; Mundy, J; Mur, L A J; Petersen, M; Smertenko, A; Taliansky, M; Van Breusegem, F; Wolpert, T; Woltering, E; Zhivotovsky, B; Bozhkov, P V

2011-08-01

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

You Shall Not Pass: Root Vacuoles as a Symplastic Checkpoint for Metal Translocation to Shoots and Possible Application to Grain Nutritional Quality

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Felipe K. Ricachenevsky

2018-04-01

Full Text Available Plant nutrient uptake is performed mostly by roots, which have to acquire nutrients while avoiding excessive amounts of essential and toxic elements. Apoplastic barriers such as the casparian strip and suberin deposition block free diffusion from the rhizosphere into the xylem, making selective plasma membrane transporters able to control elemental influx into the root symplast, efflux into the xylem and therefore shoot translocation. Additionally, transporters localized to the tonoplast of root cells have been demonstrated to regulate the shoot ionome, and may be important for seed elemental translocation. Here we review the role of vacuolar transporters in the detoxification of elements such as zinc (Zn, manganese (Mn, cadmium (Cd, cobalt (Co and nickel (Ni that are co-transported with iron (Fe during the Fe deficiency response in Arabidopsis thaliana, and the possible conservation of this mechanism in rice (Oryza sativa. We also discuss the evidence that vacuolar transporters are linked to natural variation in shoot ionome in Arabidopsis and rice, indicating that vacuolar storage might be amenable to genetic engineering without strong phenotypical changes. Finally, we discuss the possible use of root’s vacuolar transporters to increase the nutritional quality of crop grains.

Hijacking membrane transporters for arsenic phytoextraction

Science.gov (United States)

LeBlanc, Melissa S.; McKinney, Elizabeth C.; Meagher, Richard B.; Smith, Aaron P.

2012-01-01

Arsenic is a toxic metalloid and recognized carcinogen. Arsenate and arsenite are the most common arsenic species available for uptake by plants. As an inorganic phosphate (Pi) analog, arsenate is acquired by plant roots through endogenous Pi transport systems. Inside the cell, arsenate is reduced to the thiol-reactive form arsenite. Glutathione (GSH)-conjugates of arsenite may be extruded from the cell or sequestered in vacuoles by members of the ATP-binding cassette (ABC) family of transporters. In the present study we sought to enhance both plant arsenic uptake through Pi transporter overexpression, and plant arsenic tolerance through ABC transporter overexpression. We demonstrate that Arabidopsis thaliana plants overexpressing the high-affinity Pi transporter family members, AtPht1;1 or AtPht1;7, are hypersensitive to arsenate due to increased arsenate uptake. These plants do not exhibit increased sensitivity to arsenite. Co-overexpression of the yeast ABC transporter YCF1 in combination with AtPht1;1 or AtPht1;7 suppresses the arsenate-sensitive phenotype while further enhancing arsenic uptake. Taken together, our results support an arsenic transport mechanism in which arsenate uptake is increased through Pi transporter overexpression, and arsenic tolerance is enhanced through YCF1-mediated vacuolar sequestration. This work substantiates the viability of coupling enhanced uptake and vacuolar sequestration as a means for developing a prototypical engineered arsenic hyperaccumulator. PMID:23108027

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells.

Science.gov (United States)

Higaki, Takumi; Kadota, Yasuhiro; Goh, Tatsuaki; Hayashi, Teruyuki; Kutsuna, Natsumaro; Sano, Toshio; Hasezawa, Seiichiro; Kuchitsu, Kazuyuki

2008-09-01

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.

Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

International Nuclear Information System (INIS)

Kita, Ayako; Higa, Mari; Doi, Akira; Satoh, Ryosuke; Sugiura, Reiko

2015-01-01

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2 + gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis with multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2 + gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking

Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

Energy Technology Data Exchange (ETDEWEB)

Kita, Ayako; Higa, Mari [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Doi, Akira [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Satoh, Ryosuke [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Sugiura, Reiko, E-mail: sugiurar@phar.kindai.ac.jp [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan)

2015-02-13

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2{sup +} gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis with multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2{sup +} gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking.

Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals.

Science.gov (United States)

van Doorn, Wouter G; Kirasak, Kanjana; Ketsa, Saichol

2015-04-01

Prior to flower opening, mesophyll cells at the vascular bundles of Dendrobium tepals showed a large increase in vacuolar volume, partially at the expense of the cytoplasm. Electron micrographs indicated that this increase in vacuolar volume was mainly due to vacuole fusion. Macroautophagous structures typical of plant cells were observed. Only a small part of the decrease in cytoplasmic volume seemed due to macroautophagy. The vacuoles contained vesicles of various types, including multilamellar bodies. It was not clear if these vacuolar inclusions were due to macroautophagy or microautophagy. Only a single structure was observed of a protruding vacuole, indicating microautophagy. It is concluded that macroautophagy occurs in these cells but its role in vacuole formation seems small, while a possible role of microautophagy in vacuole formation might be hypothesized. Careful labeling of organelle membranes seems required to advance our insight in plant macro- and microautophagy and their roles in vacuole formation. Copyright © 2015 Elsevier GmbH. All rights reserved.

A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology.

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Florante Ricarte

Full Text Available Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface - ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY. Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes - MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

Inhibition of the Vacuolar-like ATPase from Halobacterium saccharovorum by Thiol Reagents: Evidence for Different Functional Thiols

Science.gov (United States)

Hochstein, L. I.; Stanlotter, H.; Emrich, E.; Morrison, David (Technical Monitor)

1994-01-01

N-Ethylmaleimide (NEM) inhibited the vacuolar-like ATPase from Halobacterium saccharovorum (K(sub i) approximately 1 mM) by modifying one or more of the thiols located on the largest of the subunit. ATP protected against inhibition and coincidentally prevented NEM binding which suggested that NEM acts at or near the catalytic site. p-Chloromercuriphenylsulfonate (PCMS) also inhibited this ATPase (K(sub i) approximately 90 microM). ATP did not protect against PCMS inhibition. Dithiothreitol (DTT) partially reversed PCMS inhibition and restored approximately half of the initial activity of 90% inhibited enzyme. DTT did not restore activity of the NEM-inhibited enzyme or the PCMS-inhibited enzyme when it was subsequently incubated with NEM. The failure of ATP to protect against PCMS inhibition and the inability of DTT to restore activity of enzyme incubated in the presence of PCMS and NEM suggests these reagents react with different thiols and that the PCMS-sensitive thiol may have a structural role.

Properties of the intracellular transient receptor potential (TRP) channel in yeast, Yvc1.

Science.gov (United States)

Chang, Yiming; Schlenstedt, Gabriel; Flockerzi, Veit; Beck, Andreas

2010-05-17

Transient receptor potential (TRP) channels are found among mammals, flies, worms, ciliates, Chlamydomonas, and yeast but are absent in plants. These channels are believed to be tetramers of proteins containing six transmembrane domains (TMs). Their primary structures are diverse with sequence similarities only in some short amino acid sequence motifs mainly within sequences covering TM5, TM6, and adjacent domains. In the yeast genome, there is one gene encoding a TRP-like sequence. This protein forms an ion channel in the vacuolar membrane and is therefore called Yvc1 for yeast vacuolar conductance 1. In the following we summarize its prominent features. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Subcellular localization of H(+)-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation.

Science.gov (United States)

Scherer, G F

1984-03-01

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H(+)-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H(+) pump was directly demonstrated. The H(+) pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a Δ pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K(+)-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because α-mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.

Subversion of the Endocytic and Secretory Pathways by Bacterial Effector Proteins

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Mary M. Weber

2018-01-01

Full Text Available Intracellular bacteria have developed numerous strategies to hijack host vesicular trafficking pathways to form their unique replicative niches. To promote intracellular replication, the bacteria must interact with host organelles and modulate host signaling pathways to acquire nutrients and membrane for the growing parasitophorous vacuole all while suppressing activation of the immune response. To facilitate host cell subversion, bacterial pathogens use specialized secretion systems to deliver bacterial virulence factors, termed effectors, into the host cell that mimic, agonize, and/or antagonize the function of host proteins. In this review we will discuss how bacterial effector proteins from Coxiella burnetii, Brucella abortus, Salmonella enterica serovar Typhimurium, Legionella pneumophila, Chlamydia trachomatis, and Orientia tsutsugamushi manipulate the endocytic and secretory pathways. Understanding how bacterial effector proteins manipulate host processes not only gives us keen insight into bacterial pathogenesis, but also enhances our understanding of how eukaryotic membrane trafficking is regulated.

A pivotal role of vacuolar H(+)-ATPase in regulation of lipid production in Phaeodactylum tricornutum.

Science.gov (United States)

Zhang, Huiying; Zeng, Rensen; Chen, Daoyi; Liu, Jian

2016-08-08

Microalgal lipids have been considered as a promising source for biodiesel production. Alkaline pH can induce neutral lipid accumulation in microalgae cells. However, whether and how proton pumps, especially vacuolar H(+)-ATPase (V-ATPase), function in these processes is not well known. In this study, we treated Phaeodactylum tricornutum with V-ATPase specific inhibitor bafilomycin A1 (BFA1) to determine its role in lipid production. Firstly, V-ATPase activity was increased in the latter phase of microalgae growth. BFA1 treatment decreased the cell density and lipid contents. Further analysis showed that BFA1 treatment reduced the number and size of oil bodies. GC-MS analysis showed that lipid components were not affected by BFA1 treatment. Intracellular pH was decreased and nitrogen depletion was delayed after BFA1 treatment. RNA-Seq analysis showed that expression of genes involved in calcium signaling, sulfur metabolism, cell cycle, glycolysis, pentose phosphate pathway, porphyrin, chlorophyll metabolism and lipid catabolic metabolism were upregulated, while expression of genes involved in ion transmembrane transport, ubiquitin mediated proteolysis, SNARE interactions in vesicular transport, fatty acid biosynthesis were downregulated under BFA1 treatment. Our findings provided insights into the molecular mechanisms underlying lipid accumulation and the key genes involved in lipid metabolism in Phaeodactylum tricornutum in response to BFA1.

Membranes, methods of making membranes, and methods of separating gases using membranes

Science.gov (United States)

Ho, W. S. Winston

2012-10-02

Membranes, methods of making membranes, and methods of separating gases using membranes are provided. The membranes can include at least one hydrophilic polymer, at least one cross-linking agent, at least one base, and at least one amino compound. The methods of separating gases using membranes can include contacting a gas stream containing at least one of CO.sub.2, H.sub.2S, and HCl with one side of a nonporous and at least one of CO.sub.2, H.sub.2S, and HCl selectively permeable membrane such that at least one of CO.sub.2, H.sub.2S, and HCl is selectively transported through the membrane.

The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters.

Science.gov (United States)

Cheng, Ning-Hui; Pittman, Jon K; Barkla, Bronwyn J; Shigaki, Toshiro; Hirschi, Kendal D

2003-02-01

The Arabidopsis Ca(2+)/H(+) transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca(2+) levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca(2+)/H(+) antiport activity, a 40% reduction in tonoplast V-type H(+)-translocating ATPase activity, a 36% increase in tonoplast Ca(2+)-ATPase activity, and increased expression of the putative vacuolar Ca(2+)/H(+) antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn(2+) and Mg(2+) stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters.

The Arabidopsis cax1 Mutant Exhibits Impaired Ion Homeostasis, Development, and Hormonal Responses and Reveals Interplay among Vacuolar Transporters

Science.gov (United States)

Cheng, Ning-Hui; Pittman, Jon K.; Barkla, Bronwyn J.; Shigaki, Toshiro; Hirschi, Kendal D.

2003-01-01

The Arabidopsis Ca2+/H+ transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca2+ levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca2+/H+ antiport activity, a 40% reduction in tonoplast V-type H+-translocating ATPase activity, a 36% increase in tonoplast Ca2+-ATPase activity, and increased expression of the putative vacuolar Ca2+/H+ antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn2+ and Mg2+ stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters. PMID:12566577

Application of dynamic membranes in anaerobic membranes in anaerobic membrane bioreactor systems

NARCIS (Netherlands)

Erşahin, M.E.

2015-01-01

Anaerobic membrane bioreactors (AnMBRs) physically ensure biomass retention by the application of a membrane filtration process. With growing application experiences from aerobic membrane bioreactors (MBRs), the combination of membrane and anaerobic processes has received much attention and become

Hydrolytic enzymes in the central vacuole of plant cells

International Nuclear Information System (INIS)

Boller, T.; Kende, H.

1979-01-01

The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast: (a) purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation; (b) hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation; and (c) vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: α-mannosidase, β-N-acetylglucosaminidase, β-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. None of the vacuolar enzymes investigated ws found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter

pH and Ion Homeostasis on Plant Endomembrane Dynamics: Insights from structural models and mutants of K+/H+ antiporters.

Science.gov (United States)

Sze, Heven; Chanroj, Salil

2018-04-24

Plants remodel their cells through the dynamic endomembrane system. Intracellular pH is important for membrane trafficking, but the determinants of pH homeostasis are poorly defined in plants. Electrogenic proton (H+) pumps depend on counter-ion fluxes to establish transmembrane pH gradients at the plasma membrane and endomembranes. Vacuolar-type H+-ATPase-mediated acidification of the trans-Golgi network (TGN) is crucial for secretion and membrane recycling. Pump and counter-ion fluxes are unlikely to fine-tune pH; rather, alkali cation/H+ antiporters, which can alter pH and/or cation homeostasis locally and transiently, are prime candidates. Plants have a large family of predicted cation/H+ exchangers (CHX) of obscure function, in addition to the well-studied K+(Na+)/H+ exchangers (NHX). Here, we review the regulation of cytosolic and vacuolar pH, highlighting the similarities and distinctions of NHX and CHX members. In planta, alkalinization of the TGN or vacuole by NHXs promotes membrane trafficking, endocytosis, cell expansion, and growth. CHXs localize to endomembranes and/or the plasma membrane, contribute to male fertility, pollen tube guidance, pollen wall construction, stomatal opening, and in soybean (Glycine max), tolerance to salt stress. Three-dimensional structural models and mutagenesis of Arabidopsis thaliana genes have allowed us to infer that AtCHX17 and AtNHX1 share a global architecture and a translocation core like bacterial Na+/H+ antiporters. Yet the presence of distinct residues suggests some CHXs differ from NHXs in pH sensing and electrogenicity. How H+ pumps, counter-ion fluxes, and cation/H+ antiporters are linked with signaling and membrane trafficking to remodel membranes and cell walls awaits further investigation. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

International Nuclear Information System (INIS)

Yamamoto, Masaya; Kawanabe, Mitsuyoshi; Hayashi, Yoko; Endo, Toshiya; Nishikawa, Shuh-ichi

2010-01-01

Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.

A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

Energy Technology Data Exchange (ETDEWEB)

Yamamoto, Masaya; Kawanabe, Mitsuyoshi; Hayashi, Yoko; Endo, Toshiya [Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 (Japan); Nishikawa, Shuh-ichi, E-mail: shuh@biochem.chem.nagoya-u.ac.jp [Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 (Japan)

2010-03-12

Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.

Smart membranes for monitoring membrane based desalination processes

KAUST Repository

Laleg-Kirati, Taous-Meriem

2017-10-12

Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and/or impedance sensors) mounted on the porous surface. In another example, a membrane distillation (MD) process includes the membrane. Processing circuitry can be configured to monitor outputs of the plurality of sensors. The monitored outputs can be used to determine membrane degradation, membrane fouling, or to provide an indication of membrane replacement or cleaning. The sensors can also provide temperatures or temperature differentials across the porous surface, which can be used to improve modeling or control the MD process.

Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

Directory of Open Access Journals (Sweden)

Caroline Kulangara

Full Text Available In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1. In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

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Erick Vaz Guimarães

2009-03-01

Full Text Available Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.; Choi, Seung Hak

2012-01-01

. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.

2012-06-24

Membrane reactors are generally applied in high temperature reactions (>400 °C). In the field of fine chemical synthesis, however, much milder conditions are generally applicable and polymeric membranes were applied without their damage. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane is to be used in. In this chapter a review of up to date literature about polymers and configuration catalyst/ membranes used in some recent polymeric membrane reactors is given. The new emerging concept of polymeric microcapsules as catalytic microreactors has been proposed. © 2012 Bentham Science Publishers. All rights reserved.

Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

Science.gov (United States)

Barkla, B J; Blumwald, E

1991-12-15

The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport.

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane.

Science.gov (United States)

Roz, Netta; Rehavi, Moshe

2003-06-13

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.

Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin in rice seed.

Science.gov (United States)

Kumamaru, Toshihiro; Uemura, Yuji; Inoue, Yoshimi; Takemoto, Yoko; Siddiqui, Sadar Uddin; Ogawa, Masahiro; Hara-Nishimura, Ikuko; Satoh, Hikaru

2010-01-01

To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs.

Hyaluronan synthesis in cultured tobacco cells (BY-2) expressing a chlorovirus enzyme: cytological studies.

Science.gov (United States)

Rakkhumkaew, Numfon; Shibatani, Shigeo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

2013-04-01

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured-cells (BY-2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY-2 cells harboring a vSPO-cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N-terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Copyright © 2012 Wiley Periodicals, Inc.

Membrane fouling mechanism of biofilm-membrane bioreactor (BF-MBR): Pore blocking model and membrane cleaning.

Science.gov (United States)

Zheng, Yi; Zhang, Wenxiang; Tang, Bing; Ding, Jie; Zheng, Yi; Zhang, Zhien

2018-02-01

Biofilm membrane bioreactor (BF-MBR) is considered as an important wastewater treatment technology that incorporates advantages of both biofilm and MBR process, as well as can alleviate membrane fouling, with respect to the conventional activated sludge MBR. But, to be efficient, it necessitates the establishment of proper methods for the assessment of membrane fouling. Four Hermia membrane blocking models were adopted to quantify and evaluate the membrane fouling of BF-MBR. The experiments were conducted with various operational conditions, including membrane types, agitation speeds and transmembrane pressure (TMP). Good agreement between cake formation model and experimental data was found, confirming the validity of the Hermia models for assessing the membrane fouling of BF-MBR and that cake layer deposits on membrane. Moreover, the influences of membrane types, agitation speeds and transmembrane pressure on the Hermia pore blocking coefficient of cake layer were investigated. In addition, the permeability recovery after membrane cleaning at various operational conditions was studied. This work confirms that, unlike conventional activated sludge MBR, BF-MBR possesses a low degree of membrane fouling and a higher membrane permeability recovery after cleaning. Copyright © 2017 Elsevier Ltd. All rights reserved.

pH homeostasis links the nutrient sensing PKA/TORC1/Sch9 ménage-à-trois to stress tolerance and longevity.

Science.gov (United States)

Deprez, Marie-Anne; Eskes, Elja; Wilms, Tobias; Ludovico, Paula; Winderickx, Joris

2018-01-12

The plasma membrane H + -ATPase Pma1 and the vacuolar V-ATPase act in close harmony to tightly control pH homeostasis, which is essential for a vast number of physiological processes. As these main two regulators of pH are responsive to the nutritional status of the cell, it seems evident that pH homeostasis acts in conjunction with nutrient-induced signalling pathways. Indeed, both PKA and the TORC1-Sch9 axis influence the proton pumping activity of the V-ATPase and possibly also of Pma1. In addition, it recently became clear that the proton acts as a second messenger to signal glucose availability via the V-ATPase to PKA and TORC1-Sch9. Given the prominent role of nutrient signalling in longevity, it is not surprising that pH homeostasis has been linked to ageing and longevity as well. A first indication is provided by acetic acid, whose uptake by the cell induces toxicity and affects longevity. Secondly, vacuolar acidity has been linked to autophagic processes, including mitophagy. In agreement with this, a decline in vacuolar acidity was shown to induce mitochondrial dysfunction and shorten lifespan. In addition, the asymmetric inheritance of Pma1 has been associated with replicative ageing and this again links to repercussions on vacuolar pH. Taken together, accumulating evidence indicates that pH homeostasis plays a prominent role in the determination of ageing and longevity, thereby providing new perspectives and avenues to explore the underlying molecular mechanisms.

Membrane dynamics

DEFF Research Database (Denmark)

Bendix, Pól Martin

2015-01-01

Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

Science.gov (United States)

Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

2015-08-01

Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Smart membranes for monitoring membrane based desalination processes

KAUST Repository

Laleg-Kirati, Taous-Meriem; Karam, Ayman M.

2017-01-01

Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and

Biomimetic membranes and methods of making biomimetic membranes

Science.gov (United States)

Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

2016-11-08

The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

Gel layer formation on membranes in Membrane Bioreactors

NARCIS (Netherlands)

Van den Brink, P.F.H.

2014-01-01

The widespread application of membrane bioreactors (MBRs) for municipal wastewater treatment is hampered by membrane fouling. Fouling increases energy demand, reduces process performance and creates the need for more frequent (chemical) membrane cleaning or replacement. Membrane fouling in MBRs is

Nanodisc-solubilized membrane protein library reflects the membrane proteome.

Science.gov (United States)

Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

2013-05-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

Flux Enhancement in Membrane Distillation Using Nanofiber Membranes

Directory of Open Access Journals (Sweden)

T. Jiříček

2016-01-01

Full Text Available Membrane distillation (MD is an emerging separation technology, whose largest application potential lies in the desalination of highly concentrated solutions, which are out of the scope of reverse osmosis. Despite many attractive features, this technology is still awaiting large industrial application. The main reason is the lack of commercially available membranes with fluxes comparable to reverse osmosis. MD is a thermal separation process driven by a partial vapour pressure difference. Flux, distillate purity, and thermal efficiency are always in conflict, all three being strictly connected with pore size, membrane hydrophobicity, and thickness. The world has not seen the ideal membrane yet, but nanofibers may offer a solution to these contradictory requirements. Membranes of electrospun PVDF were tested under various conditions on a direct contact (DCMD unit, in order to determine the optimum conditions for maximum flux. In addition, their performance was compared to commonly available PTFE, PE, and PES membranes. It was confirmed that thinner membranes have higher fluxes and a lower distillate purity and also higher energy losses via conduction across the membrane. As both mass and heat transfer are connected, it is best to develop new membranes with a target application in mind, for the specific membrane module and operational conditions.

Fabrication of electrospun nanofibrous membranes for membrane distillation application

KAUST Repository

Francis, Lijo

2013-02-01

Nanofibrous membranes of Matrimid have been successfully fabricated using an electrospinning technique under optimized conditions. Nanofibrous membranes are found to be highly hydrophobic with a high water contact angle of 130°. Field emission scanning electron microscopy and pore size distribution analysis revealed the big pore size structure of electrospun membranes to be greater than 2 μm and the pore size distribution is found to be narrow. Flat sheet Matrimid membranes were fabricated via casting followed by phase inversion. The morphology, pore size distribution, and water contact angle were measured and compared with the electrospun membranes. Both membranes fabricated by electrospinning and phase inversion techniques were tested in a direct contact membrane distillation process. Electrospun membranes showed high water vapor flux of 56 kg/m2-h, which is very high compared to the casted membrane as well as most of the fabricated and commercially available highly hydrophobic membranes. ©2013 Desalination Publications.

Erythrocyte remodeling in Plasmodium berghei infection: the contribution of SEP family members.

Science.gov (United States)

Currà, Chiara; Pace, Tomasino; Franke-Fayard, Blandine M D; Picci, Leonardo; Bertuccini, Lucia; Ponzi, Marta

2012-03-01

The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions. © 2011 John Wiley & Sons A/S.

Alternative energy efficient membrane bioreactor using reciprocating submerged membrane.

Science.gov (United States)

Ho, J; Smith, S; Roh, H K

2014-01-01

A novel membrane bioreactor (MBR) pilot system, using membrane reciprocation instead of air scouring, was operated at constant high flux and daily fluctuating flux to demonstrate its application under peak and diurnal flow conditions. Low and stable transmembrane pressure was achieved at 40 l/m(2)/h (LMH) by use of repetitive membrane reciprocation. The results reveal that the inertial forces acting on the membrane fibers effectively propel foulants from the membrane surface. Reciprocation of the hollow fiber membrane is beneficial for the constant removal of solids that may build up on the membrane surface and inside the membrane bundle. The membrane reciprocation in the reciprocating MBR pilot consumed less energy than coarse air scouring used in conventional MBR systems. Specific energy consumption for the membrane reciprocation was 0.072 kWh/m(3) permeate produced at 40 LMH flux, which is 75% less than for a conventional air scouring system as reported in literature without consideration of energy consumption for biological aeration (0.29 kWh/m(3)). The daily fluctuating flux test confirmed that the membrane reciprocation is effective to handle fluctuating flux up to 50 LMH. The pilot-scale reciprocating MBR system successfully demonstrated that fouling can be controlled via 0.43 Hz membrane reciprocation with 44 mm or higher amplitude.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport.

Science.gov (United States)

Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

2015-04-01

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. © 2015 Arlt et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

Science.gov (United States)

Verweij, Walter; Spelt, Cornelis E; Bliek, Mattijs; de Vries, Michel; Wit, Niek; Faraco, Marianna; Koes, Ronald; Quattrocchio, Francesca M

2016-03-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. © 2016 American Society of Plant Biologists. All rights reserved.

Disruption of a Plasmodium falciparum gene linked to male sexual development causes early arrest in gametocytogenesis.

Science.gov (United States)

Furuya, Tetsuya; Mu, Jianbing; Hayton, Karen; Liu, Anna; Duan, Junhui; Nkrumah, Louis; Joy, Deirdre A; Fidock, David A; Fujioka, Hisashi; Vaidya, Akhil B; Wellems, Thomas E; Su, Xin-zhuan

2005-11-15

A male gametocyte defect in the Plasmodium falciparum Dd2 parasite was previously discovered through the observation that all progeny clones in a Dd2 x HB3 genetic cross were the result of fertilization events between Dd2 female and HB3 male gametes. A determinant linked to the defect in Dd2 was subsequently mapped to an 800-kb segment on chromosome 12. Here, we report further mapping of the determinant to an 82-kb region and the identification of a candidate gene, P. falciparum male development gene 1 (pfmdv-1), that is expressed at a lower level in Dd2 compared with the wild-type normal male gametocyte-producing ancestor W2. Pfmdv-1 protein is sexual-stage specific and is located on the gametocyte plasma membrane, parasitophorous vacuole membrane, and the membranes of cleft-like structures within the erythrocyte. Disruption of pfmdv-1 results in a dramatic reduction in mature gametocytes, especially functional male gametocytes, with the majority of sexually committed parasites developmentally arrested at stage I. The pfmdv-1-knockout parasites show disturbed membrane structures, particularly multimembrane vesicles/tubes that likely derive from deformed cleft-like structures. Mosquito infectivity of the knockout parasites was also greatly reduced but not completely lost. The results suggest that pfmdv-1 plays a key role in gametocyte membrane formation and integrity.

Membrane order in the plasma membrane and endocytic recycling compartment.

Science.gov (United States)

Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

Science.gov (United States)

Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R; Roepe, Paul D

2016-03-31

Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

The pore-forming bacterial effector, VopQ, halts autophagic turnover.

Science.gov (United States)

Sreelatha, Anju; Orth, Kim; Starai, Vincent J

2013-12-01

Vibrio parahemolyticus Type III effector VopQ is both necessary and sufficient to induce autophagy within one hour of infection. We demonstrated that VopQ interacts with the Vo domain of the conserved vacuolar H(+)-ATPase. Membrane-associated VopQ subsequently forms pores in the membranes of acidic compartments, resulting in immediate release of protons without concomitant release of lumenal protein contents. These studies show how a bacterial pathogen can compromise host ion potentials using a gated pore-forming effector to equilibrate levels of small molecules found in endolysosomal compartments and disrupt cellular processes such as autophagy.

Giant plasma membrane vesicles: models for understanding membrane organization.

Science.gov (United States)

Levental, Kandice R; Levental, Ilya

2015-01-01

The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes

Directory of Open Access Journals (Sweden)

Glushakova Svetlana

2013-01-01

Full Text Available Abstract Background Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. Results A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions The parasite egress

Structure and physical properties of bio membranes and model membranes

International Nuclear Information System (INIS)

Tibor Hianik

2006-01-01

Bio membranes belong to the most important structures of the cell and the cell organelles. They play not only structural role of the barrier separating the external and internal part of the membrane but contain also various functional molecules, like receptors, ionic channels, carriers and enzymes. The cell membrane also preserves non-equilibrium state in a cell which is crucial for maintaining its excitability and other signaling functions. The growing interest to the bio membranes is also due to their unique physical properties. From physical point of view the bio membranes, that are composed of lipid bilayer into which are incorporated integral proteins and on their surface are anchored peripheral proteins and polysaccharides, represent liquid s crystal of smectic type. The bio membranes are characterized by anisotropy of structural and physical properties. The complex structure of bio membranes makes the study of their physical properties rather difficult. Therefore several model systems that mimic the structure of bio membranes were developed. Among them the lipid monolayers at an air-water interphase, bilayer lipid membranes, supported bilayer lipid membranes and liposomes are most known. This work is focused on the introduction into the physical word of the bio membranes and their models. After introduction to the membrane structure and the history of its establishment, the physical properties of the bio membranes and their models are stepwise presented. The most focus is on the properties of lipid monolayers, bilayer lipid membranes, supported bilayer lipid membranes and liposomes that were most detailed studied. This lecture has tutorial character that may be useful for undergraduate and graduate students in the area of biophysics, biochemistry, molecular biology and bioengineering, however it contains also original work of the author and his co-worker and PhD students, that may be useful also for specialists working in the field of bio membranes and model

Polyurethane Nanofiber Membranes for Waste Water Treatment by Membrane Distillation

Directory of Open Access Journals (Sweden)

T. Jiříček

2017-01-01

Full Text Available Self-sustained electrospun polyurethane nanofiber membranes were manufactured and tested on a direct-contact membrane distillation unit in an effort to find the optimum membrane thickness to maximize flux rate and minimize heat losses across the membrane. Also salt retention and flux at high salinities up to 100 g kg−1 were evaluated. Even though the complex structure of nanofiber layers has extreme specific surface and porosity, membrane performance was surprisingly predictable; the highest flux was achieved with the thinnest membranes and the best energy efficiency was achieved with the thickest membranes. All membranes had salt retention above 99%. Nanotechnology offers the potential to find modern solutions for desalination of waste waters, by introducing new materials with revolutionary properties, but new membranes must be developed according to the target application.

Arabidopsis Vacuolar Pyrophosphatase gene (AVP1) induces drought and salt tolerance in Nicotiana tabacum plants (abstract)

International Nuclear Information System (INIS)

Arif, A.; Mohsin, A.M.; Shafiq, S.; Zafar, Y.; Hameed, S.M.; Arif, M.; Javed, M.; Gaxiola, R.A.

2005-01-01

Drought and salinity are global problems. In Pakistan these problems are increasing to an alarming situation due to low rain-fall and bad agricultural practices. Salt and drought stress shows a high degree of similarity with respect to physiological, biochemical, molecular and genetic effects. This is due to the fact that sub-lethal salt-stress condition is ultimately an osmotic effect which is apparently similar to that brought in by water deficit. Genetic engineering allows the re-introduction of plant genes into their genomes by increasing their expression level. Plant vacuoles play a central role in cellular mechanisms of adaptation to salinity and drought stresses. In principle, increased vacuolar solute accumulation should have a positive impact in the adaptation of plants to salinity and drought. The active transport of the solutes depends on the proton gradients established by proton pumps. We have over expressed Arabidopsis gene AVP1 (Arabidopsis thaliana vacuolar pyro phosphatase H/sup +/ pump) to increase drought/salt tolerance in tobacco. The AVP1 ORF with a tandem repeat of 358 promoter was cloned in pPZP212 vector and Agrobacterium-mediated transformation was performed. Transgenic plants were selected on plant nutrient agar medium supplemented with 50 mg/liter kanamycin. Transgenic plants were confirmed for transfer of genes by AVP1 and nptll gene specific PCR and Southern hybridization. AVP1 transgenic plants were screened for salt tolerance by providing NaCl solution in addition to nutrient solution. AVP1 transgenic plants showed tolerance up to 300 mM NaCl as compared to control which died ten days after 200 mM NaCl. Sodium and potassium were measured in salt treated and control plants. Results showed that sodium ion uptake in the salt treated transgenic plants was four times more as compared to wild type. This remarkable increase in Na/sup +/ ion uptake indicates that AVP1 vacuole proton pumps are actively involved in the transport of Na

Cation trapping by cellular acidic compartments: Beyond the concept of lysosomotropic drugs

Energy Technology Data Exchange (ETDEWEB)

Marceau, François, E-mail: francois.marceau@crchul.ulaval.ca [Centre de recherche en rhumatologie et immunologie, Centre Hospitalier Universitaire de Québec, Québec QC, Canada G1V 4G2 (Canada); Bawolak, Marie-Thérèse [Centre de recherche en rhumatologie et immunologie, Centre Hospitalier Universitaire de Québec, Québec QC, Canada G1V 4G2 (Canada); Lodge, Robert [Centre de recherche en infectiologie, Centre Hospitalier Universitaire de Québec, Québec QC, Canada G1V 4G2 (Canada); Bouthillier, Johanne; Gagné-Henley, Angélique [Centre de recherche en rhumatologie et immunologie, Centre Hospitalier Universitaire de Québec, Québec QC, Canada G1V 4G2 (Canada); Gaudreault, René C. [Unité des Biotechnologies et de Bioingénierie, Centre Hospitalier Universitaire de Québec, Québec QC, Canada G1L 3L5 (Canada); Morissette, Guillaume [Centre de recherche en rhumatologie et immunologie, Centre Hospitalier Universitaire de Québec, Québec QC, Canada G1V 4G2 (Canada)

2012-02-15

“Lysosomotropic” cationic drugs are known to concentrate in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping); they draw water by an osmotic mechanism, leading to a vacuolar response. Several aspects of this phenomenon were recently reexamined. (1) The proton pump vacuolar (V)-ATPase is the driving force of cationic drug uptake and ensuing vacuolization. In quantitative transport experiments, V-ATPase inhibitors, such as bafilomycin A1, greatly reduced the uptake of cationic drugs and released them in preloaded cells. (2) Pigmented or fluorescent amines are effectively present in a concentrated form in the large vacuoles. (3) Consistent with V-ATPase expression in trans-Golgi, lysosomes and endosomes, a fraction of the vacuoles is consistently labeled with trans-Golgi markers and protein secretion and endocytosis are often inhibited in vacuolar cells. (4) Macroautophagic signaling (accumulation of lipidated and membrane-bound LC3 II) and labeling of the large vacuoles by the autophagy effector LC3 were consistently observed in cells, precisely at incubation periods and amine concentrations that cause vacuolization. Vacuoles also exhibit late endosome/lysosome markers, because they may originate from such organelles or because macroautophagosomes fuse with lysosomes. Autophagosome persistence is likely due to the lack of resolution of autophagy, rather than to nutritional deprivation. (5) Increased lipophilicity decreases the threshold concentration for the vacuolar and autophagic cytopathology, because simple diffusion into cells is limiting. (6) A still unexplained mitotic arrest is consistently observed in cells loaded with amines. An extended recognition of relevant clinical situations is proposed for local or systemic drug administration.

pH homeostasis links the nutrient sensing PKA/TORC1/Sch9 ménage-à-trois to stress tolerance and longevity

Directory of Open Access Journals (Sweden)

Marie-Anne Deprez

2018-01-01

Full Text Available The plasma membrane H+-ATPase Pma1 and the vacuolar V-ATPase act in close harmony to tightly control pH homeostasis, which is essential for a vast number of physiological processes. As these main two regulators of pH are responsive to the nutritional status of the cell, it seems evident that pH homeostasis acts in conjunction with nutrient-induced signalling pathways. Indeed, both PKA and the TORC1-Sch9 axis influence the proton pumping activity of the V-ATPase and possibly also of Pma1. In addition, it recently became clear that the proton acts as a second messenger to signal glucose availability via the V-ATPase to PKA and TORC1-Sch9. Given the prominent role of nutrient signalling in longevity, it is not surprising that pH homeostasis has been linked to ageing and longevity as well. A first indication is provided by acetic acid, whose uptake by the cell induces toxicity and affects longevity. Secondly, vacuolar acidity has been linked to autophagic processes, including mitophagy. In agreement with this, a decline in vacuolar acidity was shown to induce mitochondrial dysfunction and shorten lifespan. In addition, the asymmetric inheritance of Pma1 has been associated with replicative ageing and this again links to repercussions on vacuolar pH. Taken together, accumulating evidence indicates that pH homeostasis plays a prominent role in the determination of ageing and longevity, thereby providing new perspectives and avenues to explore the underlying molecular mechanisms.

Cation trapping by cellular acidic compartments: Beyond the concept of lysosomotropic drugs

International Nuclear Information System (INIS)

Marceau, François; Bawolak, Marie-Thérèse; Lodge, Robert; Bouthillier, Johanne; Gagné-Henley, Angélique; Gaudreault, René C.; Morissette, Guillaume

2012-01-01

“Lysosomotropic” cationic drugs are known to concentrate in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping); they draw water by an osmotic mechanism, leading to a vacuolar response. Several aspects of this phenomenon were recently reexamined. (1) The proton pump vacuolar (V)-ATPase is the driving force of cationic drug uptake and ensuing vacuolization. In quantitative transport experiments, V-ATPase inhibitors, such as bafilomycin A1, greatly reduced the uptake of cationic drugs and released them in preloaded cells. (2) Pigmented or fluorescent amines are effectively present in a concentrated form in the large vacuoles. (3) Consistent with V-ATPase expression in trans-Golgi, lysosomes and endosomes, a fraction of the vacuoles is consistently labeled with trans-Golgi markers and protein secretion and endocytosis are often inhibited in vacuolar cells. (4) Macroautophagic signaling (accumulation of lipidated and membrane-bound LC3 II) and labeling of the large vacuoles by the autophagy effector LC3 were consistently observed in cells, precisely at incubation periods and amine concentrations that cause vacuolization. Vacuoles also exhibit late endosome/lysosome markers, because they may originate from such organelles or because macroautophagosomes fuse with lysosomes. Autophagosome persistence is likely due to the lack of resolution of autophagy, rather than to nutritional deprivation. (5) Increased lipophilicity decreases the threshold concentration for the vacuolar and autophagic cytopathology, because simple diffusion into cells is limiting. (6) A still unexplained mitotic arrest is consistently observed in cells loaded with amines. An extended recognition of relevant clinical situations is proposed for local or systemic drug administration.

Fouling in Membrane Distillation, Osmotic Distillation and Osmotic Membrane Distillation

Directory of Open Access Journals (Sweden)

Mourad Laqbaqbi

2017-03-01

Full Text Available Various membrane separation processes are being used for seawater desalination and treatment of wastewaters in order to deal with the worldwide water shortage problem. Different types of membranes of distinct morphologies, structures and physico-chemical characteristics are employed. Among the considered membrane technologies, membrane distillation (MD, osmotic distillation (OD and osmotic membrane distillation (OMD use porous and hydrophobic membranes for production of distilled water and/or concentration of wastewaters for recovery and recycling of valuable compounds. However, the efficiency of these technologies is hampered by fouling phenomena. This refers to the accumulation of organic/inorganic deposits including biological matter on the membrane surface and/or in the membrane pores. Fouling in MD, OD and OMD differs from that observed in electric and pressure-driven membrane processes such electrodialysis (ED, membrane capacitive deionization (MCD, reverse osmosis (RO, nanofiltration (NF, ultrafiltration (UF, microfiltration (MF, etc. Other than pore blockage, fouling in MD, OD and OMD increases the risk of membrane pores wetting and reduces therefore the quantity and quality of the produced water or the concentration efficiency of the process. This review deals with the observed fouling phenomena in MD, OD and OMD. It highlights different detected fouling types (organic fouling, inorganic fouling and biofouling, fouling characterization techniques as well as various methods of fouling reduction including pretreatment, membrane modification, membrane cleaning and antiscalants application.

Flow and fouling in membrane filters: Effects of membrane morphology

Science.gov (United States)

Sanaei, Pejman; Cummings, Linda J.

2015-11-01

Membrane filters are widely-used in microfiltration applications. Many types of filter membranes are produced commercially, for different filtration applications, but broadly speaking the requirements are to achieve fine control of separation, with low power consumption. The answer to this problem might seem obvious: select the membrane with the largest pore size and void fraction consistent with the separation requirements. However, membrane fouling (an inevitable consequence of successful filtration) is a complicated process, which depends on many parameters other than membrane pore size and void fraction; and which itself greatly affects the filtration process and membrane functionality. In this work we formulate mathematical models that can (i) account for the membrane internal morphology (internal structure, pore size & shape, etc.); (ii) fouling of membranes with specific morphology; and (iii) make some predictions as to what type of membrane morphology might offer optimum filtration performance.

Host and bacterial proteins that repress recruitment of LC3 to Shigella early during infection.

Directory of Open Access Journals (Sweden)

Leigh A Baxt

Full Text Available Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min., the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4-6 hr by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG. Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.

The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells.

Directory of Open Access Journals (Sweden)

Karen A Conrad

Full Text Available The ubiquitous presence of SPFH (Stomatin, Prohibitin, Flotillin, HflK/HflC proteins in all domains of life suggests that their function would be conserved. However, SPFH functions are diverse with organism-specific attributes. SPFH proteins play critical roles in physiological processes such as mechanosensation and respiration. Here, we characterize the stomatin ORF19.7296/SLP3 in the opportunistic human pathogen Candida albicans. Consistent with the localization of stomatin proteins, a Slp3p-Yfp fusion protein formed visible puncta along the plasma membrane. We also visualized Slp3p within the vacuolar lumen. Slp3p primary sequence analyses identified four putative S-palmitoylation sites, which may facilitate membrane localization and are conserved features of stomatins. Plasma membrane insertion sequences are present in mammalian and nematode SPFH proteins, but are absent in Slp3p. Strikingly, Slp3p was present in yeast cells, but was absent in hyphal cells, thus categorizing it as a yeast-phase specific protein. Slp3p membrane fluorescence significantly increased in response to cellular stress caused by plasma membrane, cell wall, oxidative, or osmotic perturbants, implicating SLP3 as a general stress-response gene. A slp3Δ/Δ homozygous null mutant had no detected phenotype when slp3Δ/Δ mutants were grown in the presence of a variety of stress agents. Also, we did not observe a defect in ion accumulation, filamentation, endocytosis, vacuolar structure and function, cell wall structure, or cytoskeletal structure. However, SLP3 over-expression triggered apoptotic-like death following prolonged exposure to oxidative stress or when cells were induced to form hyphae. Our findings reveal the cellular localization of Slp3p, and for the first time associate Slp3p function with the oxidative stress response.

Evasion of the Immune Response by Trypanosoma cruzi during Acute Infection

Science.gov (United States)

Cardoso, Mariana S.; Reis-Cunha, João Luís; Bartholomeu, Daniella C.

2016-01-01

Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical disease that affects millions of people mainly in Latin America. To establish a life-long infection, T. cruzi must subvert the vertebrate host’s immune system, using strategies that can be traced to the parasite’s life cycle. Once inside the vertebrate host, metacyclic trypomastigotes rapidly invade a wide variety of nucleated host cells in a membrane-bound compartment known as the parasitophorous vacuole, which fuses to lysosomes, originating the phagolysosome. In this compartment, the parasite relies on a complex network of antioxidant enzymes to shield itself from lysosomal oxygen and nitrogen reactive species. Lysosomal acidification of the parasitophorous vacuole is an important factor that allows trypomastigote escape from the extremely oxidative environment of the phagolysosome to the cytoplasm, where it differentiates into amastigote forms. In the cytosol of infected macrophages, oxidative stress instead of being detrimental to the parasite, favors amastigote burden, which then differentiates into bloodstream trypomastigotes. Trypomastigotes released in the bloodstream upon the rupture of the host cell membrane express surface molecules, such as calreticulin and GP160 proteins, which disrupt initial and key components of the complement pathway, while others such as glycosylphosphatidylinositol-mucins stimulate immunoregulatory receptors, delaying the progression of a protective immune response. After an immunologically silent entry at the early phase of infection, T. cruzi elicits polyclonal B cell activation, hypergammaglobulinemia, and unspecific anti-T. cruzi antibodies, which are inefficient in controlling the infection. Additionally, the coexpression of several related, but not identical, epitopes derived from trypomastigote surface proteins delays the generation of T. cruzi-specific neutralizing antibodies. Later in the infection, the establishment of an anti-T. cruzi

Evasion of the immune response by Trypanosoma cruzi during acute infection

Directory of Open Access Journals (Sweden)

Mariana Santos Cardoso

2016-01-01

Full Text Available Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical disease that affects millions of people mainly in Latin America. To establish a life-long infection, T. cruzi must subvert the vertebrate host’s immune system, using strategies that can be traced to the parasite’s life cycle. Once inside the vertebrate host, metacyclic trypomastigotes rapidly invade a wide variety of nucleated host cells in a membrane-bound compartment known as the parasitophorous vacuole, which fuses to lysosomes, originating the phagolysosome. In this compartment, the parasite relies on a complex network of antioxidant enzymes to shield itself from lysosomal oxygen and nitrogen reactive species. Lysosomal acidification of the parasitophorous vacuole is an important factor that allows trypomastigote escape from the extremely oxidative environment of the phagolysosome to the cytoplasm, where it differentiates into amastigote forms. In the cytosol of infected macrophages, oxidative stress instead of being detrimental to the parasite, favors amastigote burden, which then differentiates into bloodstream trypomastigotes. Trypomastigotes released in the bloodstream upon the rupture of the host cell membrane express surface molecules, such as calreticulin and GP160 proteins, which disrupt initial and key components of the complement pathway, while others such as GPI-mucins stimulate immunoregulatory receptors, delaying the progression of a protective immune response. After an immunologically silent entry at the early phase of infection, T. cruzi elicits polyclonal B cell activation, hypergammaglobulinemia, and unspecific anti-T. cruzi antibodies, which are inefficient in controlling the infection. Additionally, the co-expression of several related but not identical epitopes derived from trypomastigote surface proteins delays the generation of T. cruzi-specific neutralizing antibodies. Later in the infection, the establishment of an anti-T. cruzi CD8

Characteristics of weak base-induced vacuoles formed around individual acidic organelles.

Science.gov (United States)

Hiruma, Hiromi; Kawakami, Tadashi

2011-01-01

We have previously found that the weak base 4-aminopyridine induces Brownian motion of acidic organelles around which vacuoles are formed, causing organelle traffic disorder in neurons. Our present study investigated the characteristics of vacuoles induced by weak bases (NH(4)Cl, aminopyridines, and chloroquine) using mouse cells. Individual vacuoles included acidic organelles identified by fluorescent protein expression. Mitochondria and actin filaments were extruded outside the vacuoles, composing the vacuole rim. Staining with amine-reactive fluorescence showed no protein/amino acid content in vacuoles. Thus, serous vacuolar contents are probably partitioned by viscous cytosol, other organelles, and cytoskeletons, but not membrane. The weak base (chloroquine) was immunochemically detected in intravacuolar organelles, but not in vacuoles. Early vacuolization was reversible, but long-term vacuolization caused cell death. The vacuolization and cell death were blocked by the vacuolar H(+)-ATPase inhibitor and Cl--free medium. Staining with LysoTracker or LysoSensor indicated that intravacuolar organelles were strongly acidic and vacuoles were slightly acidic. This suggests that vacuolization is caused by accumulation of weak base and H(+) in acidic organelles, driven by vacuolar H(+)-ATPase associated with Cl(-) entering, and probably by subsequent extrusion of H(+) and water from organelles to the surrounding cytoplasm.

Fundamentals of membrane bioreactors materials, systems and membrane fouling

CERN Document Server

Ladewig, Bradley

2017-01-01

This book provides a critical, carefully researched, up-to-date summary of membranes for membrane bioreactors. It presents a comprehensive and self-contained outline of the fundamentals of membrane bioreactors, especially their relevance as an advanced water treatment technology. This outline helps to bring the technology to the readers’ attention, and positions the critical topic of membrane fouling as one of the key impediments to its more widescale adoption. The target readership includes researchers and industrial practitioners with an interest in membrane bioreactors.

Adaptive silicone-membrane lenses: planar vs. shaped membrane

CSIR Research Space (South Africa)

Schneider, F

2009-08-01

Full Text Available Engineering, Georges-Koehler-Allee 102, Freiburg 79110, Germany florian.schneider@imtek.uni-freiburg.de ABSTRACT We compare the performance and optical quality of two types of adaptive fluidic silicone-membrane lenses. The membranes feature either a...-membrane lenses: planar vs. shaped membrane Florian Schneider1,2, Philipp Waibel2 and Ulrike Wallrabe2 1 CSIR, Materials Science and Manufacturing, PO Box 395, Pretoria 0001, South Africa 2 University of Freiburg – IMTEK, Department of Microsystems...

Degradation of Polypropylene Membranes Applied in Membrane Distillation Crystallizer

Directory of Open Access Journals (Sweden)

Marek Gryta

2016-03-01

Full Text Available The studies on the resistance to degradation of capillary polypropylene membranes assembled in a membrane crystallizer were performed. The supersaturation state of salt was achieved by evaporation of water from the NaCl saturated solutions using membrane distillation process. A high feed temperature (363 K was used in order to enhance the degradation effects and to shorten the test times. Salt crystallization was carried out by the application of batch or fluidized bed crystallizer. A significant membrane scaling was observed regardless of the method of realized crystallization. The SEM-EDS, DSC, and FTIR methods were used for investigations of polypropylene degradation. The salt crystallization onto the membrane surface accelerated polypropylene degradation. Due to a polymer degradation, the presence of carbonyl groups on the membranes’ surface was identified. Besides the changes in the chemical structure a significant mechanical damage of the membranes, mainly caused by the internal scaling, was also found. As a result, the membranes were severely damaged after 150 h of process operation. A high level of salt rejection was maintained despite damage to the external membrane surface.

Membrane fusion

DEFF Research Database (Denmark)

Bendix, Pól Martin

2015-01-01

At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

Introducing Membrane Charge and Membrane Potential to T Cell Signaling

Directory of Open Access Journals (Sweden)

Yuanqing Ma

2017-11-01

Full Text Available While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins, and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR–CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.

Polyazole hollow fiber membranes for direct contact membrane distillation

KAUST Repository

Maab, Husnul; Alsaadi, Ahmad Salem; Francis, Lijo; Livazovic, Sara; Ghaffour, NorEddine; Amy, Gary L.; Nunes, Suzana Pereira

2013-01-01

Porous hollow fiber membranes were fabricated from fluorinated polyoxadiazole and polytriazole by a dry-wet spinning method for application in desalination of Red Sea water by direct contact membrane distillation (DCMD). The data were compared with commercially available hollow fiber MD membranes prepared from poly(vinylidene fluoride). The membranes were characterized by electron microscopy, liquid entry pressure (LEP), and pore diameter measurements. Finally, the hollow fiber membranes were tested for DCMD. Salt selectivity as high as 99.95% and water fluxes as high as 35 and 41 L m -2 h-1 were demonstrated, respectively, for polyoxadiazole and polytriazole hollow fiber membranes, operating at 80 C feed temperature and 20 C permeate. © 2013 American Chemical Society.

Polyazole hollow fiber membranes for direct contact membrane distillation

KAUST Repository

Maab, Husnul

2013-08-07

Porous hollow fiber membranes were fabricated from fluorinated polyoxadiazole and polytriazole by a dry-wet spinning method for application in desalination of Red Sea water by direct contact membrane distillation (DCMD). The data were compared with commercially available hollow fiber MD membranes prepared from poly(vinylidene fluoride). The membranes were characterized by electron microscopy, liquid entry pressure (LEP), and pore diameter measurements. Finally, the hollow fiber membranes were tested for DCMD. Salt selectivity as high as 99.95% and water fluxes as high as 35 and 41 L m -2 h-1 were demonstrated, respectively, for polyoxadiazole and polytriazole hollow fiber membranes, operating at 80 C feed temperature and 20 C permeate. © 2013 American Chemical Society.

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

Directory of Open Access Journals (Sweden)

Rita Marcia Cardoso de Paiva

2015-12-01

Full Text Available Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy.

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Masahito Asada

Full Text Available BACKGROUND: Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed "gliding motility". However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs, and gliding motility has so far not been observed in the parasite. METHODOLOGY/PRINCIPAL FINDINGS: Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion. CONCLUSIONS/SIGNIFICANCE: This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

Uptake and intracellular fate of [14C]sucrose-insulin in perfused rat livers

International Nuclear Information System (INIS)

Surmacz, C.A.; Wert, J.J. Jr.; Ward, W.F.; Mortimore, G.E.

1988-01-01

Insulin was covalently linked to [ 14 C]sucrose by means of cyanuric chloride to provide a label that would remain entrapped within the vacuolar system. The uptake of the conjugate by the perfused rat liver was rapid, competitively inhibited by native insulin, and abolished by alkali denaturation. As assessed by its distribution on self-generating gradients of colloidal silica-povidone, label in lysosome-enriched samples of liver taken at different times after the addition of the conjugate moved progressively during 15 min from the plasma membrane into an intermediate peak and then to dense lysosomal fractions. After 30-60 min, the label had equilibrated throughout the lysosomal-vacuolar system. The initial movement from the plasma membrane to the intermediate peak occurred between 2 and 5 min. Because label in the peak could be physically separated from the lysosomal marker, β-acetylglucosaminidase, by dispersing the sample through the gradient mixture before centrifugation rather than layering it, the authors concluded that the intermediate particles in question were not lysosomal in nature. On gel-filtration chromatography, label extracted from the intermediate peak did not move with insulin but rather as a broad band of lower molecular weight products, suggesting that insulin is subject to early proteolytic attack within a nonlysosomal compartment

Uptake and intracellular fate of [14C]sucrose-insulin in perfused rat livers.

Science.gov (United States)

Surmacz, C A; Wert, J J; Ward, W F; Mortimore, G E

1988-07-01

Insulin was covalently linked to [14C]sucrose by means of cyanuric chloride to provide a label that would remain entrapped within the vacuolar system. The uptake of the conjugate by the perfused rat liver was rapid (half-life = 2.9 min), competitively inhibited by native insulin, and abolished by alkali denaturation. As assessed by its distribution on self-generating gradients of colloidal silica-povidone, label in lysosome-enriched samples of liver taken at different times after the addition of the conjugate moved progressively during 15 min from the plasma membrane into an intermediate peak and then to dense lysosomal fractions. After 30-60 min, the label had equilibrated throughout the lysosomal-vacuolar system. The initial movement from the plasma membrane to the intermediate peak occurred between 2 and 5 min. Because label in the peak could be physically separated from the lysosomal marker, beta-acetylglucosaminidase, by dispersing the sample through the gradient mixture before centrifugation rather than layering it, we concluded that the intermediate particles in question were not lysosomal in nature. On gel-filtration chromatography, label extracted from the intermediate peak did not move with insulin but rather as a broad band of lower molecular weight products, suggesting that insulin is subject to early proteolytic attack within a nonlysosomal compartment.

Vfa1 Binds to the N-terminal Microtubule-interacting and Trafficking (MIT) Domain of Vps4 and Stimulates Its ATPase Activity*

Science.gov (United States)

Vild, Cody J.; Xu, Zhaohui

2014-01-01

The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function. PMID:24567329

Vfa1 binds to the N-terminal microtubule-interacting and trafficking (MIT) domain of Vps4 and stimulates its ATPase activity.

Science.gov (United States)

Vild, Cody J; Xu, Zhaohui

2014-04-11

The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

Science.gov (United States)

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Liver plasma membranes: an effective method to analyze membrane proteome.

Science.gov (United States)

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

Membrane Biophysics

CERN Document Server

Ashrafuzzaman, Mohammad

2013-01-01

Physics, mathematics and chemistry all play a vital role in understanding the true nature and functioning of biological membranes, key elements of living processes. Besides simple spectroscopic observations and electrical measurements of membranes we address in this book the phenomena of coexistence and independent existence of different membrane components using various theoretical approaches. This treatment will be helpful for readers who want to understand biological processes by applying both simple observations and fundamental scientific analysis. It provides a deep understanding of the causes and effects of processes inside membranes, and will thus eventually open new doors for high-level pharmaceutical approaches towards fighting membrane- and cell-related diseases.

Polyurethane Nanofiber Membranes for Waste Water Treatment by Membrane Distillation

OpenAIRE

Jiříček, T.; Komárek, M.; Lederer, T.

2017-01-01

Self-sustained electrospun polyurethane nanofiber membranes were manufactured and tested on a direct-contact membrane distillation unit in an effort to find the optimum membrane thickness to maximize flux rate and minimize heat losses across the membrane. Also salt retention and flux at high salinities up to 100 g kg−1 were evaluated. Even though the complex structure of nanofiber layers has extreme specific surface and porosity, membrane performance was surprisingly predictable; the highest ...

Nanodisc-solubilized membrane protein library reflects the membrane proteome

OpenAIRE

Marty, Michael T.; Wilcox, Kyle C.; Klein, William L.; Sligar, Stephen G.

2013-01-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membr...

Impact of sludge flocs on membrane fouling in membrane bioreactors

DEFF Research Database (Denmark)

Christensen, Morten Lykkegaard; Niessen, Wolfgang; Jørgensen, Mads Koustrup

Membrane bioreactors (MBR) are widely used for wastewater treatment, but membrane fouling reduces membrane performance and thereby increases the cost for membranes and fouling control. Large variation in filtration properties measured as flux decline was observed for the different types of sludges....... Further, the flux could partly be reestablished after the relaxation period depending on the sludge composition. The results underline that sludge properties are important for membrane fouling and that control of floc properties, as determined by the composition of the microbial communities...... and the physico-chemical properties, is an efficient method to reduce membrane fouling in the MBR. High concentration of suspended extracellular substances (EPS) and small particles (up to 10 µm) resulted in pronounced fouling propensity. The membrane fouling resistance was reduced at high concentration...

Model for Stress-induced Protein Degradation in Lemna minor1

Science.gov (United States)

Cooke, Robert J.; Roberts, Keith; Davies, David D.

1980-01-01

Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins. PMID:16661588

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

Science.gov (United States)

Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

2017-09-01

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

Pretreatment and Membrane Hydrophilic Modification to Reduce Membrane Fouling

Directory of Open Access Journals (Sweden)

Huaqiang Chu

2013-09-01

Full Text Available The application of low pressure membranes (microfiltration/ultrafiltration has undergone accelerated development for drinking water production. However, the major obstacle encountered in its popularization is membrane fouling caused by natural organic matter (NOM. This paper firstly summarizes the two factors causing the organic membrane fouling, including molecular weight (MW and hydrophilicity/hydrophobicity of NOM, and then presents a brief introduction of the methods which can prevent membrane fouling such as pretreatment of the feed water (e.g., coagulation, adsorption, and pre-oxidation and membrane hydrophilic modification (e.g., plasma modification, irradiation grafting modification, surface coating modification, blend modification, etc.. Perspectives of further research are also discussed.

Recent developments on ion-exchange membranes and electro-membrane processes.

Science.gov (United States)

Nagarale, R K; Gohil, G S; Shahi, Vinod K

2006-02-28

Rapid growth of chemical and biotechnology in diversified areas fuels the demand for the need of reliable green technologies for the down stream processes, which include separation, purification and isolation of the molecules. Ion-exchange membrane technologies are non-hazardous in nature and being widely used not only for separation and purification but their application also extended towards energy conversion devices, storage batteries and sensors etc. Now there is a quite demand for the ion-exchange membrane with better selectivities, less electrical resistance, high chemical, mechanical and thermal stability as well as good durability. A lot of work has been done for the development of these types of ion-exchange membranes during the past twenty-five years. Herein we have reviewed the preparation of various types of ion-exchange membranes, their characterization and applications for different electro-membrane processes. Primary attention has been given to the chemical route used for the membrane preparation. Several general reactions used for the preparation of ion-exchange membranes were described. Methodologies used for the characterization of these membranes and their applications were also reviewed for the benefit of readers, so that they can get all information about the ion-exchange membranes at one platform. Although there are large number of reports available regarding preparations and applications of ion-exchange membranes more emphasis were predicted for the usefulness of these membranes or processes for solving certain type of industrial or social problems. More efforts are needed to bring many products or processes to pilot scale and extent their applications.

The Coxiella Burnetii type IVB secretion system (T4BSS) component DotA is released/secreted during infection of host cells and during in vitro growth in a T4BSS-dependent manner.

Science.gov (United States)

Luedtke, Brandon E; Mahapatra, Saugata; Lutter, Erika I; Shaw, Edward I

2017-06-01

Coxiella burnetii is a Gram-negative intracellular pathogen and is the causative agent of the zoonotic disease Q fever. To cause disease, C. burnetii requires a functional type IVB secretion system (T4BSS) to transfer effector proteins required for the establishment and maintenance of a membrane-bound parasitophorous vacuole (PV) and further modulation of host cell process. However, it is not clear how the T4BSS interacts with the PV membrane since neither a secretion pilus nor an extracellular pore forming apparatus has not been described. To address this, we used the acidified citrate cysteine medium (ACCM) along with cell culture infection and immunological techniques to identify the cellular and extracellular localization of T4BSS components. Interestingly, we found that DotA and IcmX were secreted/released in a T4BSS-dependent manner into the ACCM. Analysis of C. burnetii-infected cell lines revealed that DotA colocalized with the host cell marker CD63 (LAMP3) at the PV membrane. In the absence of bacterial protein synthesis, DotA also became depleted from the PV membrane. These data are the first to identify the release/secretion of C. burnetii T4BSS components during axenic growth and the interaction of a T4BSS component with the PV membrane during infection of host cells. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Clustering on Membranes

DEFF Research Database (Denmark)

Johannes, Ludger; Pezeshkian, Weria; Ipsen, John H

2018-01-01

Clustering of extracellular ligands and proteins on the plasma membrane is required to perform specific cellular functions, such as signaling and endocytosis. Attractive forces that originate in perturbations of the membrane's physical properties contribute to this clustering, in addition to direct...... protein-protein interactions. However, these membrane-mediated forces have not all been equally considered, despite their importance. In this review, we describe how line tension, lipid depletion, and membrane curvature contribute to membrane-mediated clustering. Additional attractive forces that arise...... from protein-induced perturbation of a membrane's fluctuations are also described. This review aims to provide a survey of the current understanding of membrane-mediated clustering and how this supports precise biological functions....

Identification of a new target of miR-16, Vacuolar Protein Sorting 4a.

Directory of Open Access Journals (Sweden)

Neeta Adhikari

Full Text Available The rationale was to utilize a bioinformatics approach to identify miRNA binding sites in genes with single nucleotide mutations (SNPs to discover pathways in heart failure (HF.The objective was to focus on the genes containing miRNA binding sites with miRNAs that were significantly altered in end-stage HF and in response to a left ventricular assist device (LVAD.BEDTools v2.14.3 was used to discriminate SNPs within predicted 3'UTR miRNA binding sites. A member of the miR-15/107 family, miR-16, was decreased in the circulation of end-stage HF patients and increased in response to a LVAD (p<0.001. MiR-16 decreased Vacuolar Protein Sorting 4a (VPS4a expression in HEK 293T cells (p<0.01. The SNP rs16958754 was identified in the miR-15/107 family binding site of VPS4a which abolished direct binding of miR-16 to the 3'UTR of VPS4a (p<0.05. VPS4a was increased in the circulation of end-stage HF patients (p<0.001, and led to a decrease in the number of HEK 293T cells in vitro (p<0.001.We provide evidence that miR-16 decreases in the circulation of end-stage HF patients and increases with a LVAD. Modeling studies suggest that miR-16 binds to and decreases expression of VPS4a. Overexpression of VPS4a decreases cell number. Together, these experiments suggest that miR-16 and VPS4a expression are altered in end-stage HF and in response to unloading with a LVAD. This signaling pathway may lead to reduced circulating cell number in HF.

Analysis of proton exchange membrane fuel cell performance with alternate membranes

Energy Technology Data Exchange (ETDEWEB)

Wakizoe, Masanobu; Velev, O A; Srinivasan, S [Texas A and M Univ., College Station, TX (United States). Texas Engineering Experiment Station

1995-02-01

Renewed interest in proton exchange membrane fuel cell technology for space and terrestrial (particularly electric vehicles) was stimulated by the demonstration, in the mid 1980s, of high energy efficiencies and high power densities. One of the most vital components of the PEMFC is the proton conducting membrane. In this paper, an analysis is made of the performances of PEMFCs with Dupont`s Nafion, Dow`s experimental, and Asahi Chemical`s Aciplex-S membranes. Attempts were also made to draw correlations between the PEMFC performances with the three types of membranes and their physico-chemical characteristics. Practically identical levels of performances (energy efficiency, power density, and lifetime) were achieved in PEMFCs with the Dow and the Aciplex-S membranes and these performances were better than in the PEMFCs with the Nafion-115 membrane. The electrode kinetic parameters for oxygen reduction are better for the PEMFCs with the Aciplex-S and Nafion membranes than with the Dow membranes. The PEMFCs with the Aciplex-S and Dow membranes have nearly the same internal resistances which are considerably lower than for the PEMFC with the Nafion membrane. The desired membrane characteristics to obtain high levels of performance are low equivalent weight and high water content. (Author)

Hydrolytic enzymes in the central vacuole of plant cells.

Science.gov (United States)

Boller, T; Kende, H

1979-06-01

The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. (a) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. (b) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. (c) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated.THE INTRACELLULAR ACTIVITIES OF THE FOLLOWING ACID HYDROLASES WERE PRIMARILY LOCALIZED IN THE VACUOLE OF TOBACCO CELLS: alpha-mannosidase, beta-N-acetylglucosaminidase, beta-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. Our data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome.None of the vacuolar enzymes investigated was found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar

Electrospun superhydrophobic membranes with unique structures for membrane distillation.

Science.gov (United States)

Liao, Yuan; Loh, Chun-Heng; Wang, Rong; Fane, Anthony G

2014-09-24

With modest temperature demand, low operating pressure, and high solute rejection, membrane distillation (MD) is an attractive option for desalination, waste treatment, and food and pharmaceutical processing. However, large-scale practical applications of MD are still hindered by the absence of effective membranes with high hydrophobicity, high porosity, and adequate mechanical strength, which are important properties for MD permeation fluxes, stable long-term performance, and effective packing in modules without damage. This study describes novel design strategies for highly robust superhydrophobic dual-layer membranes for MD via electrospinning. One of the newly developed membranes comprises a durable and ultrathin 3-dimensional (3D) superhydrophobic skin and porous nanofibrous support whereas another was fabricated by electrospinning 3D superhydrophobic layers on a nonwoven support. These membranes exhibit superhydrophobicity toward distilled water, salty water, oil-in-water emulsion, and beverages, which enables them to be used not only for desalination but also for other processes. The superhydrophobic dual-layer membrane #3S-N with nanofibrous support has a competitive permeation flux of 24.6 ± 1.2 kg m(-2) h(-1) in MD (feed and permeate temperate were set as 333 and 293 K, respectively) due to the higher porosity of the nanofibrous scaffold. Meanwhile, the membranes with the nonwoven support exhibit greater mechanical strength due to this support combined with better long-term performance because of the thicker 3D superhydrophobic layers. The morphology, pore size, porosity, mechanical properties, and liquid enter pressure of water of these superhydrophobic composite membranes with two different structures are reported and compared with commercial polyvinylidene fluoride membranes.

Microporous silica membranes

DEFF Research Database (Denmark)

Boffa, Vittorio; Yue, Yuanzheng

2012-01-01

Hydrothermal stability is a crucial factor for the application of microporous silica-based membranes in industrial processes. Indeed, it is well established that steam exposure may cause densification and defect formation in microporous silica membranes, which are detrimental to both membrane...... permeability and selectivity. Numerous previous studies show that microporous transition metal doped-silica membranes are hydrothermally more stable than pure silica membranes, but less permeable. Here we present a quantitative study on the impact of type and concentration of transition metal ions...... on the microporous structure, stability and permeability of amorphous silica-based membranes, providing information on how to design chemical compositions and synthetic paths for the fabrication of silica-based membranes with a well accessible and highly stabile microporous structure....

Composite Membrane with Underwater-Oleophobic Surface for Anti-Oil-Fouling Membrane Distillation.

Science.gov (United States)

Wang, Zhangxin; Hou, Deyin; Lin, Shihong

2016-04-05

In this study, we fabricated a composite membrane for membrane distillation (MD) by modifying a commercial hydrophobic polyvinylidene fluoride (PVDF) membrane with a nanocomposite coating comprising silica nanoparticles, chitosan hydrogel and fluoro-polymer. The composite membrane exhibits asymmetric wettability, with the modified surface being in-air hydrophilic and underwater oleophobic, and the unmodified surface remaining hydrophobic. By comparing the performance of the composite membrane and the pristine PVDF membrane in direct contact MD experiments using a saline emulsion with 1000 ppm crude oil (in water), we showed that the fabricated composite membrane was significantly more resistant to oil fouling compared to the pristine hydrophobic PVDF membrane. Force spectroscopy was conducted for the interaction between an oil droplet and the membrane surface using a force tensiometer. The difference between the composite membrane and the pristine PVDF membrane in their interaction with an oil droplet served to explain the difference in the fouling propensities between these two membranes observed in MD experiments. The results from this study suggest that underwater oleophobic coating can effectively mitigate oil fouling in MD operations, and that the fabricated composite membrane with asymmetric wettability can enable MD to desalinate hypersaline wastewater with high concentrations of hydrophobic contaminants.

Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6.

Science.gov (United States)

Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

2015-03-27

The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

The Loss of Vacuolar Protein Sorting 11 (vps11) Causes Retinal Pathogenesis in a Vertebrate Model of Syndromic Albinism

Science.gov (United States)

Thomas, Jennifer L.; Vihtelic, Thomas S.; denDekker, Aaron D.; Willer, Gregory; Luo, Xixia; Murphy, Taylor R.; Gregg, Ronald G.; Hyde, David R.

2011-01-01

Purpose. To establish the zebrafish platinum mutant as a model for studying vision defects caused by syndromic albinism diseases such as Chediak-Higashi syndrome, Griscelli syndrome, and Hermansky-Pudlak syndrome (HPS). Methods. Bulked segregant analysis and candidate gene sequencing revealed that the zebrafish platinum mutation is a single-nucleotide insertion in the vps11 (vacuolar protein sorting 11) gene. Expression of vps11 was determined by RT-PCR and in situ hybridization. Mutants were analyzed for pigmentation defects and retinal disease by histology, immunohistochemistry, and transmission electron microscopy. Results. Phenocopy and rescue experiments determined that a loss of Vps11 results in the platinum phenotype. Expression of vps11 appeared ubiquitous during zebrafish development, with stronger expression in the developing retina and retinal pigmented epithelium (RPE). Zebrafish platinum mutants exhibited reduced pigmentation in the body and RPE; however, melanophore development, migration, and dispersion occurred normally. RPE, photoreceptors, and inner retinal neurons formed normally in zebrafish platinum mutants. However, a gradual loss of RPE, an absence of mature melanosomes, and the subsequent degradation of RPE/photoreceptor interdigitation was observed. Conclusions. These data show that Vps11 is not necessary for normal retinal development or initiation of melanin biosynthesis, but is essential for melanosome maturation and healthy maintenance of the RPE and photoreceptors. PMID:21330665

Comparison and analysis of membrane fouling between flocculent sludge membrane bioreactor and granular sludge membrane bioreactor.

Directory of Open Access Journals (Sweden)

Wang Jing-Feng

Full Text Available The goal of this study is to investigate the effect of inoculating granules on reducing membrane fouling. In order to evaluate the differences in performance between flocculent sludge and aerobic granular sludge in membrane reactors (MBRs, two reactors were run in parallel and various parameters related to membrane fouling were measured. The results indicated that specific resistance to the fouling layer was five times greater than that of mixed liquor sludge in the granular MBR. The floc sludge more easily formed a compact layer on the membrane surface, and increased membrane resistance. Specifically, the floc sludge had a higher moisture content, extracellular polymeric substances concentration, and negative surface charge. In contrast, aerobic granules could improve structural integrity and strength, which contributed to the preferable permeate performance. Therefore, inoculating aerobic granules in a MBR presents an effective method of reducing the membrane fouling associated with floc sludge the perspective of from the morphological characteristics of microbial aggregates.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

OpenAIRE

Evan Quon; Christopher T. Beh

2016-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer...

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

Science.gov (United States)

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Metal–Organic Framework-Functionalized Alumina Membranes for Vacuum Membrane Distillation

Directory of Open Access Journals (Sweden)

Jian Zuo

2016-12-01

Full Text Available Nature-mimetic hydrophobic membranes with high wetting resistance have been designed for seawater desalination via vacuum membrane distillation (VMD in this study. This is achieved through molecular engineering of metal–organic framework (MOF-functionalized alumina surfaces. A two-step synthetic strategy was invented to design the hydrophobic membranes: (1 to intergrow MOF crystals on the alumina tube substrate and (2 to introduce perfluoro molecules onto the MOF functionalized membrane surface. With the first step, the surface morphology, especially the hierarchical roughness, can be controlled by tuning the MOF crystal structure. After the second step, the perfluoro molecules function as an ultrathin layer of hydrophobic floss, which lowers the surface energy. Therefore, the resultant membranes do not only possess the intrinsic advantages of alumina supports such as high stability and high water permeability, but also have a hydrophobic surface formed by MOF functionalization. The membrane prepared under an optimum condition achieved a good VMD flux of 32.3 L/m2-h at 60 °C. This study may open up a totally new approach for design of next-generation high performance membrane distillation membranes for seawater desalination.

Mechanisms of invasion from sporozoite and merozoíto of Plasmodium

Directory of Open Access Journals (Sweden)

Lilian M. Spencer

2016-05-01

Full Text Available Malaria or paludismo is caused in humans by four species of Plasmodium belonging to phylum Apicomplexa: ovale, malaria, vivax and falciparum, being the last, the responsible of the clinical complication and death in the vertebrate host. Plasmodium parasite possess a specialized secretory organelles called rhoptries, micronemes and dense granules that facilitate invasion of host cells. The sporozoite stage of Plasmodium travels through the different cells of vertebrate host until it reaches the hepatocyte and have been form the parasitophorous vacuole. The infected hepatocytes rupture, results in the releasing thousands of daughter merozoites that invade the erythrocytes with the formation of parasitophorous vacuole too. Several researchers suggest the gliding motility mechanism as the responsible of hepatocyte invasion. While, which the erythrocyte invasion process has been described as the result of tree steps: first contact, re-orientation and invasion. In this review the surface proteins of merozoites and esporozoites are pointed out as the most important factors for the molecular invasion mechanisms until the elaboration of the parasitophorous vacuole. These proteins that take part in these mechanisms are the possible candidates in the design of an anti-malaria vaccine.

Focus on Membrane Differentiation and Membrane Domains in the Prokaryotic Cell

NARCIS (Netherlands)

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

Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

Science.gov (United States)

Sun, Bingyun; Hood, Leroy

2014-06-06

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

Chemical degradation mechanisms of membranes for alkaline membrane fuel cells

Energy Technology Data Exchange (ETDEWEB)

Choe, Yoong-Kee [National Institute of Advanced Industrial Science and Technology, Umezono 1-1-1, Tsukuba (Japan); Henson, Neil J.; Kim, Yu Seung [Los Alamos National Laboratory, Los Alamos, NM (United States)

2015-12-31

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

Nafion/Silicon Oxide Composite Membrane for High Temperature Proton Exchange Membrane Fuel Cell

Institute of Scientific and Technical Information of China (English)

无

2007-01-01

Nafion/Silicon oxide composite membranes were produced via in situ sol-gel reaction of tetraethylorthosilicate (TEOS) in Nafion membranes. The physicochemical properties of the membranes were studied by FT-IR, TG-DSC and tensile strength. The results show that the silicon oxide is compatible with the Nafion membrane and the thermo stability of Nafion/Silicon oxide composite membrane is higher than that of Nafion membrane. Furthermore, the tensile strength of Nafion/Silicon oxide composite membrane is similar to that of the Nafion membrane. The proton conductivity of Nafion/Silicon oxide composite membrane is higher than that of Nafion membrane. When the Nafion/Silicon oxide composite membrane was employed as an electrolyte in H2/O2 PEMFC, a higher current density value (1 000 mA/cm2 at 0.38 V) than that of the Nafion 1135 membrane (100 mA/cm2 at 0.04 V) was obtained at 110 ℃.

The LRRC8A Mediated “Swell Activated” Chloride Conductance Is Dispensable for Vacuolar Homeostasis in Neutrophils

Directory of Open Access Journals (Sweden)

Philippe Behe

2017-05-01

Full Text Available The dialysis of human and mouse neutrophils in patch clamp experiments in the conventional whole-cell mode induces the emergence of a chloride (Cl- current that appeared to be primarily regulated by cytoplasmic ionic strength. The characteristics of this current resembled that of the classical, and ubiquitous volume-sensitive outwardly rectifying Cl- current: strong outward rectification, selectivity sequence of the Eisenman1 type, insensitivity to external pH and strong inhibition by tamoxifen, DCPIB and WW781. We show that this current is essentially supported by the leucine rich repeat containing 8 A (LRRC8A; the naturally occurring LRRC8A truncation mutant in ebo/ebo mice drastically reduced Cl- conductance in neutrophils. Remarkably, the residual component presents a distinct pharmacology, but appears equally potentiated by reduced ionic strength. We have investigated the role of the LRRC8A-supported current in the ionic homeostasis of the phagosomal compartment. The vacuolar pH, measured using SNARF-1 labeled Candida albicans, normally rises because of NADPH oxidase activity, and this elevation is blocked by certain Cl- channel inhibitors. However, the pH rise remains intact in neutrophils from the ebo/ebo mice which also demonstrate preserved phagocytic and respiratory burst capacities and normal-sized vacuoles. Thus, the LRRC8A-dependent conductance of neutrophils largely accounts for their “swell activated” Cl- current, but is not required for homeostasis of the phagosomal killing compartment.

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

Science.gov (United States)

Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

2014-10-01

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

Eggshell membrane-templated porous gold membranes using nanoparticles as building blocks

International Nuclear Information System (INIS)

Ashraf, S.; Khalid, Z. M.; Hussain, I.

2013-01-01

Highly porous gold membrane-like structures are formed using eggshell membrane, as such and heat denatured, as a template and gold nanoparticles as building blocks. Gold nanoparticles were produced in-situ on the eggshell membranes without using additional reducing agents. The morphology and loading of gold nanoparticles can easily be controlled by adjusting the pH and thus the redox potential of eggshell membranes. Lower pH favored the formation of irregularly-shaped but dense gold macro/ nanocrystals whereas higher pH(8-9) favored the formation of fairly uniform but less dense gold nanoparticles onto the eggshell membranes. Heat treatment of eggshell membrane-gold nanoparticle composites formed at pH 8-9 led to the formation of highly porous membrane like gold while mimicking the original structure of eggshell membrane. All these materials have been thoroughly characterized using field emission scanning electron microscopy (FESEM), energy dispersive X-ray spectroscopy (EDX), and inductively coupled plasma - atomic emission spectroscopy (ISP-AES). These highly porous membrane-like gold materials may have potential applications in catalysis, biosensors, electrode materials, optically selective coatings, heat dissipation and biofiltration. (author)

A plant-like proton-pump partnership in the malaria parasite

International Nuclear Information System (INIS)

Allen, R.J.W.; Saliba, K.J.; Zissis, S.; Kirk, K.

2001-01-01

Full text: The 'intraerythrocytic' form of the human malaria parasite. Plasmodium falciparum contains an acidic 'digestive vacuole' which is believed to be the main site of haemoglobin degradation, and the major site of action of many antimalarial drugs. The mechanism/s by which this organelle is acidified have not been investigated. In plant cells, the internal acidic vacuole has on its membrane two types of H + -pumps which contribute to the generation of an acidic pH: a vacuolar-type H + -ATPase (V-H + -ATPase) and a vacuolar H + -pyrophosphatase (V-H + -PPase). The presence of a V-H + -ATPase on the digestive vacuole membrane of P. falciparum has been demonstrated by immuno-electron microscopy (J. Biol. Chem. (2000) 275: 34353-34358) but its functional activity on this organelle has not been demonstrated. Two V-H + -PPase genes have been shown to be expressed in the intraerythrocytic stage of the P. falciparum parasite (Mol. Biochem. Parasitol. (2001) 114: 183-195); however, immunological methods failed to detect either on the parasite digestive vacuole. In this study we use a combination of NMR spectroscopy and fluorescence techniques to show that (i) P. falciparum contains low levels of pyrophosphate, and (ii) that both ATP and pyrophosphate are able to energise the acidification of the parasite's digestive vacuole. We propose that, like many plant cells the digestive vacuole of P. falciparum parasites has, on its membrane, a V-H + -PPase as well as a V-H + -ATPaSe, and that both pumps contribute to the pH regulation of this organelle

Membrane processes

Science.gov (United States)

Staszak, Katarzyna

2017-11-01

The membrane processes have played important role in the industrial separation process. These technologies can be found in all industrial areas such as food, beverages, metallurgy, pulp and paper, textile, pharmaceutical, automotive, biotechnology and chemical industry, as well as in water treatment for domestic and industrial application. Although these processes are known since twentieth century, there are still many studies that focus on the testing of new membranes' materials and determining of conditions for optimal selectivity, i. e. the optimum transmembrane pressure (TMP) or permeate flux to minimize fouling. Moreover the researchers proposed some calculation methods to predict the membrane processes properties. In this article, the laboratory scale experiments of membrane separation techniques, as well their validation by calculation methods are presented. Because membrane is the "heart" of the process, experimental and computational methods for its characterization are also described.

Polymeric and Lipid Membranes-From Spheres to Flat Membranes and vice versa.

Science.gov (United States)

Saveleva, Mariia S; Lengert, Ekaterina V; Gorin, Dmitry A; Parakhonskiy, Bogdan V; Skirtach, Andre G

2017-08-15

Membranes are important components in a number of systems, where separation and control of the flow of molecules is desirable. Controllable membranes represent an even more coveted and desirable entity and their development is considered to be the next step of development. Typically, membranes are considered on flat surfaces, but spherical capsules possess a perfect "infinite" or fully suspended membranes. Similarities and transitions between spherical and flat membranes are discussed, while applications of membranes are also emphasized.

Chelating polymeric membranes

KAUST Repository

Peinemann, Klaus-Viktor; Villalobos Vazquez de la Parra, Luis Francisco; Hilke, Roland

2015-01-01

microporous chelating polymeric membrane. Embodiments include, but are not limited to, microporous chelating polymeric membranes, device comprising the membranes, and methods of using and making the same.

Photoresponsive nanostructured membranes

KAUST Repository

Madhavan, Poornima

2016-07-26

The perspective of adding stimuli-response to isoporous membranes stimulates the development of separation devices with pores, which would open or close under control of environment chemical composition, temperature or exposure to light. Changes in pH and temperature have been previously investigated. In this work, we demonstrate for the first time the preparation of photoresponsive isoporous membranes, applying self-assembly non-solvent induced phase separation to a new light responsive block copolymer. First, we optimized the membrane formation by using poly(styrene-b-anthracene methyl methacrylate-b-methylmethacrylate) (PS-b-PAnMMA-b-PMMA) copolymer, identifying the most suitable solvent, copolymer block length, and other parameters. The obtained final triblock copolymer membrane morphologies were characterized using atomic force and electron microscopy. The microscopic analysis reveals that the PS-b-PAnMMA-b-PMMA copolymer can form both lamellar and ordered hexagonal nanoporous structures on the membrane top layer in appropriate solvent compositions. The nanostructured membrane emits fluorescence due to the presence of the anthracene mid-block. On irradiation of light the PS-b-PAnMMA-b-PMMA copolymer membranes has an additional stimuli response. The anthracene group undergoes conformational changes by forming [4 + 4] cycloadducts and this alters the membrane\\'s water flux and solute retention. © 2016 The Royal Society of Chemistry.

Prion Protein on Astrocytes or in Extracellular Fluid Impedes Neurodegeneration Induced by Truncated Prion Protein

OpenAIRE

Race, Brent; Meade-White, Kimberly; Race, Richard; Baumann, Frank; Aguzzi, Adriano; Chesebro, Bruce

2009-01-01

Prion protein (PrP) is a host-encoded membrane-anchored glycoprotein which is required for susceptibility to prion disease. PrP may also be important for normal brain functions such as hippocampal spatial memory. Previously transgenic mice expressing amino terminally truncated mouse PrP (Δ32–134) spontaneously developed a fatal disease associated with degeneration of cerebellar granular neurons as well as vacuolar degeneration of deep cerebellar and brain stem white matter. This disease could...

Recent advances on membranes and membrane reactors for hydrogen production

NARCIS (Netherlands)

Gallucci, F.; Fernandez Gesalaga, E.; Corengia, P.; Sint Annaland, van M.

2013-01-01

Membranes and membrane reactors for pure hydrogen production are widely investigated not only because of the important application areas of hydrogen, but especially because mechanically and chemically stable membranes with high perm-selectivity towards hydrogen are available and are continuously

Performance of different hollow fiber membranes for seawater desalination using membrane distillation

KAUST Repository

Francis, Lijo; Ghaffour, NorEddine; Alsaadi, Ahmad Salem; Amy, Gary L.

2014-01-01

Membrane distillation requires a highly porous hydrophobic membrane with low surface energy. In this paper, we compare the direct contact membrane distillation (DCMD) performances of four different types of in-house fabricated hollow fiber membranes and two different commercially available hollow fiber membranes. Hollow fiber membranes are fabricated using wet-jet phase inversion technique and the polymeric matrices used for the fabrication are polyvinylidine fluoride (PVDF) and polyvinyl chloride (PVC). Commercial hollow fiber membrane materials are made of polytetrafluoroethylene (PTFE) and polypropylene (PP). PVDF hollow fibers showed a superior performance among all the hollow fibers tested in the DCMD process and gave a water vapor flux of 31 kg m-2h-1 at a feed and coolant inlet temperatures of 80 and 20°C, respectively. Under the same conditions, the water vapor flux observed for PP, PTFE, and PVC hollow fiber membranes are 13, 11, and 6 kg m-2h-1, respectively, with 99.99% salt rejection observed for all membranes used.

Performance of different hollow fiber membranes for seawater desalination using membrane distillation

KAUST Repository

Francis, Lijo

2014-08-11

Membrane distillation requires a highly porous hydrophobic membrane with low surface energy. In this paper, we compare the direct contact membrane distillation (DCMD) performances of four different types of in-house fabricated hollow fiber membranes and two different commercially available hollow fiber membranes. Hollow fiber membranes are fabricated using wet-jet phase inversion technique and the polymeric matrices used for the fabrication are polyvinylidine fluoride (PVDF) and polyvinyl chloride (PVC). Commercial hollow fiber membrane materials are made of polytetrafluoroethylene (PTFE) and polypropylene (PP). PVDF hollow fibers showed a superior performance among all the hollow fibers tested in the DCMD process and gave a water vapor flux of 31 kg m-2h-1 at a feed and coolant inlet temperatures of 80 and 20°C, respectively. Under the same conditions, the water vapor flux observed for PP, PTFE, and PVC hollow fiber membranes are 13, 11, and 6 kg m-2h-1, respectively, with 99.99% salt rejection observed for all membranes used.

Improving Nanofiber Membrane Characteristics and Membrane Distillation Performance of Heat-Pressed Membranes via Annealing Post-Treatment

Directory of Open Access Journals (Sweden)

Minwei Yao

2017-01-01

Full Text Available Electrospun membranes are gaining interest for use in membrane distillation (MD due to their high porosity and interconnected pore structure; however, they are still susceptible to wetting during MD operation because of their relatively low liquid entry pressure (LEP. In this study, post-treatment had been applied to improve the LEP, as well as its permeation and salt rejection efficiency. The post-treatment included two continuous procedures: heat-pressing and annealing. In this study, annealing was applied on the membranes that had been heat-pressed. It was found that annealing improved the MD performance as the average flux reached 35 L/m2·h or LMH (>10% improvement of the ones without annealing while still maintaining 99.99% salt rejection. Further tests on LEP, contact angle, and pore size distribution explain the improvement due to annealing well. Fourier transform infrared spectroscopy and X-ray diffraction analyses of the membranes showed that there was an increase in the crystallinity of the polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP membrane; also, peaks indicating the α phase of polyvinylidene fluoride (PVDF became noticeable after annealing, indicating some β and amorphous states of polymer were converted into the α phase. The changes were favorable for membrane distillation as the non-polar α phase of PVDF reduces the dipolar attraction force between the membrane and water molecules, and the increase in crystallinity would result in higher thermal stability. The present results indicate the positive effect of the heat-press followed by an annealing post-treatment on the membrane characteristics and MD performance.

Membrane paradigm

International Nuclear Information System (INIS)

Price, R.H.; Thorne, K.S.

1986-01-01

The membrane paradigm is a modified frozen star approach to modeling black holes, with particles and fields assuming a complex, static, boundary-layer type structure (membrane) near the event horizon. The membrane has no effects on the present or future evolution of particles and fields above itself. The mathematical representation is a combination of a formalism containing terms for the shear and bulk viscosity, surface pressure, momentum, temperature, entropy, etc., of the horizon and the 3+1 formalism. The latter model considers a family of three-dimensional spacelike hypersurfaces in one-dimensional time. The membrane model considers a magnetic field threading the hole and undergoing torque from the hole rotation. The field is cleaned by the horizon and distributed over the horizon so that ohmic dissipation is minimized. The membrane paradigm is invalid inside the horizon, but is useful for theoretically probing the properties of slowly evolving black holes

Ion-conducting membranes

Science.gov (United States)

Masel, Richard I.; Sajjad, Syed Dawar; Gao, Yan; Liu, Zengcai; Chen, Qingmei

2017-12-26

An anion-conducting polymeric membrane comprises a terpolymer of styrene, vinylbenzyl-R.sub.s and vinylbenzyl-R.sub.x. R.sub.s is a positively charged cyclic amine group. R.sub.x is at least one constituent selected from the group consisting Cl, OH and a reaction product between an OH or Cl and a species other than a simple amine or a cyclic amine. The total weight of the vinylbenzyl-R.sub.x groups is greater than 0.3% of the total weight of the membrane. In a preferred embodiment, the membrane is a Helper Membrane that increases the faradaic efficiency of an electrochemical cell into which the membrane is incorporated, and also allows product formation at lower voltages than in cells without the Helper Membrane.

Influence of membrane properties on fouling in submerged membrane bioreactors

NARCIS (Netherlands)

van der Marel, P.; Zwijnenburg, A.; Kemperman, Antonius J.B.; Wessling, Matthias; Temmink, Hardy; van der Meer, Walterus Gijsbertus Joseph

2010-01-01

Polymeric flat-sheet membranes with different properties were used in filtration experiments with activated sludge from a pilot-scale MBR to investigate the influence of membrane pore size, surface porosity, pore morphology, and hydrophobicity on membrane fouling. An improved flux-step method was

Exploring the potential of commercial polyethylene membranes for desalination by membrane distillation

KAUST Repository

Zuo, Jian; Bonyadi, Sina; Chung, Neal Tai-Shung

2015-01-01

The potential of utilizing polyethylene (PE) membranes in membrane distillation (MD) for sea water desalination has been explored in this study. The advantages of using PE membranes are (1) their intrinsic hydrophobicity with low surface energy of 28-33×10N/m, (2) good chemical stability and low thermal conductivity and (3) their commercial availability that may expedite the MD commercialization process. Several commercial PE membranes with different physicochemical properties are employed to study the capability and feasibility of PE membrane application in an MD process. The effect of membrane pore size, porosity, thickness and wetting resistance on MD performance and energy efficiency have been investigated. The PE membranes demonstrate impressive separation performance with permeation fluxes reaching 123.0L/mh for a 3.5wt% sodium chloride (NaCl) feed solution at 80°C. This superior performance surpasses most of the prior commercial and lab-made flat sheet and hollow fiber membranes. A long term MD testing of 100h is also performed to evaluate the durability of PE membranes, and a relatively stable performance is observed during the entire experiment. This long term stability signifies the suitability of PE membranes for MD applications.

Exploring the potential of commercial polyethylene membranes for desalination by membrane distillation

KAUST Repository

Zuo, Jian

2015-09-26

The potential of utilizing polyethylene (PE) membranes in membrane distillation (MD) for sea water desalination has been explored in this study. The advantages of using PE membranes are (1) their intrinsic hydrophobicity with low surface energy of 28-33×10N/m, (2) good chemical stability and low thermal conductivity and (3) their commercial availability that may expedite the MD commercialization process. Several commercial PE membranes with different physicochemical properties are employed to study the capability and feasibility of PE membrane application in an MD process. The effect of membrane pore size, porosity, thickness and wetting resistance on MD performance and energy efficiency have been investigated. The PE membranes demonstrate impressive separation performance with permeation fluxes reaching 123.0L/mh for a 3.5wt% sodium chloride (NaCl) feed solution at 80°C. This superior performance surpasses most of the prior commercial and lab-made flat sheet and hollow fiber membranes. A long term MD testing of 100h is also performed to evaluate the durability of PE membranes, and a relatively stable performance is observed during the entire experiment. This long term stability signifies the suitability of PE membranes for MD applications.

Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

Science.gov (United States)

Laible, Philip D; Hanson, Deborah K

2013-06-04

The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

Micro-and/or nano-scale patterned porous membranes, methods of making membranes, and methods of using membranes

KAUST Repository

Wang, Xianbin; Chen, Wei; Wang, Zhihong; Zhang, Xixiang; Yue, Weisheng; Lai, Zhiping

2015-01-01

Embodiments of the present disclosure provide for materials that include a pre-designed patterned, porous membrane (e.g., micro- and/or nano-scale patterned), structures or devices that include a pre-designed patterned, porous membrane, methods of making pre-designed patterned, porous membranes, methods of separation, and the like.

Micro-and/or nano-scale patterned porous membranes, methods of making membranes, and methods of using membranes

KAUST Repository

Wang, Xianbin

2015-01-22

Embodiments of the present disclosure provide for materials that include a pre-designed patterned, porous membrane (e.g., micro- and/or nano-scale patterned), structures or devices that include a pre-designed patterned, porous membrane, methods of making pre-designed patterned, porous membranes, methods of separation, and the like.

Separation membrane development

Energy Technology Data Exchange (ETDEWEB)

Lee, M.W. [Savannah River Technology Center, Aiken, SC (United States)

1998-08-01

A ceramic membrane has been developed to separate hydrogen from other gases. The method used is a sol-gel process. A thin layer of dense ceramic material is coated on a coarse ceramic filter substrate. The pore size distribution in the thin layer is controlled by a densification of the coating materials by heat treatment. The membrane has been tested by permeation measurement of the hydrogen and other gases. Selectivity of the membrane has been achieved to separate hydrogen from carbon monoxide. The permeation rate of hydrogen through the ceramic membrane was about 20 times larger than Pd-Ag membrane.

Structure and properties of cell membranes. Volume 3: Methodology and properties of membranes

International Nuclear Information System (INIS)

Benga, G.

1985-01-01

This book covers the topics: Quantum chemical approach to study the mechanisms of proton translocation across membranes through protein molecules; monomolecular films as biomembrane models; planar lipid bilayers in relation to biomembranes; relation of liposomes to cell membranes; reconstitution of membrane transport systems; structure-function relationships in cell membranes as revealed by X-ray techniques; structure-function relationships in cell membranes as revealed by spin labeling ESR; structure and dynamics of cell membranes as revealed by NMR techniques; the effect of dietary lipids on the composition and properties of biological membranes and index

A forced-flow membrane reactor for transfructosylation using ceramic membrane.

Science.gov (United States)

Nishizawa, K; Nakajima, M; Nabetani, H

2000-04-05

A forced-flow membrane reactor system for transfructosylation was investigated using several ceramic membranes having different pore sizes. beta-Fructofuranosidase from Aspergillus niger ATCC 20611 was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-coupling reagent. Sucrose solution was forced through the ceramic membrane by crossflow filtration while transfructosylation took place. The saccharide composition of the product, which was a mixture of fructooligosaccharides (FOS), was a function of the permeate flux, which was easily controlled by pressure. Using 0.2 micrometer pore size of symmetric ceramic membrane, the volumetric productivity obtained was 3.87 kg m(-3) s(-1), which was 560 times higher than that in a reported batch system, with a short residence time of 11 s. The half-life of the immobilized enzyme in the membrane was estimated to be 35 days by a long-term operation. Copyright 2000 John Wiley & Sons, Inc.

Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi.

Science.gov (United States)

Malinsky, Jan; Opekarová, Miroslava; Grossmann, Guido; Tanner, Widmar

2013-01-01

The existence of specialized microdomains in plasma membranes, postulated for almost 25 years, has been popularized by the concept of lipid or membrane rafts. The idea that detergent-resistant membranes are equivalent to lipid rafts, which was generally abandoned after a decade of vigorous data accumulation, contributed to intense discussions about the validity of the raft concept. The existence of membrane microdomains, meanwhile, has been verified by unequivocal independent evidence. This review summarizes the current state of research in plants and fungi with respect to common aspects of both kingdoms. In these organisms, principally immobile microdomains large enough for microscopic detection have been visualized. These microdomains are found in the context of cell-cell interactions (plant symbionts and pathogens), membrane transport, stress, and polarized growth, and the data corroborate at least three mechanisms of formation. As documented in this review, modern methods of visualization of lateral membrane compartments are also able to uncover the functional relevance of membrane microdomains.

Toxoplasma gondii Infection Is Associated with Mitochondrial Dysfunction in-Vitro

Directory of Open Access Journals (Sweden)

Genevieve Syn

2017-12-01

Full Text Available Upon invasion of host cells, the ubiquitous pathogen Toxoplasma gondii manipulates several host processes, including re-organization of host organelles, to create a replicative niche. Host mitochondrial association to T. gondii parasitophorous vacuoles is rapid and has roles in modulating host immune responses. Here gene expression profiling of T. gondii infected cells reveals enrichment of genes involved in oxidative phosphorylation (OXPHOS and mitochondrial dysfunction 6 h post-infection. We identified 11 hub genes (HIF-1α, CASP8, FN1, POU5F1, CD44, ISG15, HNRNPA1, MDM2, RPL35, VHL, and NUPR1 and 10 predicted upstream regulators, including 4 endogenous regulators RICTOR, KDM5A, RB1, and D-glucose. We characterized a number of mitochondrial parameters in T. gondii infected human foreskin fibroblast cells over a 36 h time-course. In addition to the usual rapid recruitment and apparent enlargement of mitochondria around the parasitophorous vacuole we observed fragmented host mitochondria in infected cells, not linked to cellular apoptosis, from 24 h post-infection. An increase in mitochondrial superoxide levels in T. gondii infected cells was observed that required active parasite invasion and peaked at 30 h post-infection. Measurement of OXPHOS proteins showed decreased expression of Complex IV in infected cells at 24 h post-infection, followed by decreased expression of Complexes I and II at 36 h post-infection. No change occurred in Complex V. No difference in host mitochondrial membrane potential between infected and mock-infected cells was observed at any time. Our results show perturbation of host mitochondrial function following T. gondii infection that likely impacts on pathogenesis of disease.

Reprogramming neutral lipid metabolism in mouse dendritic leucocytes hosting live Leishmania amazonensis amastigotes.

Directory of Open Access Journals (Sweden)

Hervé Lecoeur

Full Text Available BACKGROUND: After loading with live Leishmania (L amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i long-chain fatty acids (LCFA and cholesterol uptake/transport, (ii LCFA and cholesterol (re-esterification to triacyl-sn-glycerol (TAG and cholesteryl esters (CE, respectively. As these neutral lipids are known to make up the lipid body (LB core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. CONCLUSIONS/SIGNIFICANCE: As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin?

Modification of chitosan membranes with nanosilica particles as polymer electrolyte membranes

Energy Technology Data Exchange (ETDEWEB)

Kusumastuti, Ella, E-mail: ella.kusuma@gmail.com; Siniwi, Widasari Trisna, E-mail: wsiniwi@gmail.com; Mahatmanti, F. Widhi; Jumaeri [Department of Chemistry, Faculty of Mathematics and Natural Sciences, State University of Semarang D6 Building 2" n" d floor, Sekaran Unnes Campus, Gunungpati, Semarang (Indonesia); Atmaja, Lukman; Widiastuti, Nurul [Department of Chemistry, Faculty of Mathematics and Natural Sciences, Tenth November Institute of Technology Keputih ITS Campus, Sukolilo, Surabaya (Indonesia)

2016-04-19

Chitosan has been widely used as polymer matrix for Polymer Electrolyte Membrane (PEM) application replacing Nafion which has shortcomings in terms of high methanol permeability that degrades the performance of fuel cells. Chitosan membranes modification is performed by adding nanosilica to prevent methanol transport through the membrane. Nanosilica is synthesized by sol-gel method and the particle diameter is obtained by analysis using Breunner Emmet Teller (BET) that is 6.59 nm. Nanosilica is mixed with chitosan solution to obtain nanosilica-chitosan as polymer electrolyte membrane. The membranes are synthesized through phase inversion method with nanosilica composition including 0; 0.5; 1; 2; 3; 5; and 10% w/w of chitosan. Characterization of the membranes indicate that the results of water swelling, proton conductivity and methanol permeability of the membrane with 3% nanosilica respectively were 49.23%, 0.231 S/cm, and 5.43 x 10{sup −7} cm{sup 2}/s. Based on the results of membrane selectivity calculation, the optimum membrane is the composition of 3% nanosilica with value 5.91 x 105 S s cm{sup −3}. The results of functional groups analysis with FTIR showed that it was only physical interaction that occurred between chitosan and nanosilica since no significant changes found in peak around the wave number 1000-1250 cm{sup −-1}.

Functionalization of a Hydrophilic Commercial Membrane Using Inorganic-Organic Polymers Coatings for Membrane Distillation

Directory of Open Access Journals (Sweden)

Lies Eykens

2017-06-01

Full Text Available Membrane distillation is a thermal separation technique using a microporous hydrophobic membrane. One of the concerns with respect to the industrialization of the technique is the development of novel membranes. In this paper, a commercially available hydrophilic polyethersulfone membrane with a suitable structure for membrane distillation was modified using available hydrophobic coatings using ORMOCER® technology to obtain a hydrophobic membrane that can be applied in membrane distillation. The surface modification was performed using a selection of different components, concentrations, and application methods. The resulting membranes can have two hydrophobic surfaces or a hydrophobic and hydrophilic surface depending on the application method. An extensive characterization procedure confirmed the suitability of the coating technique and the obtained membranes for membrane distillation. The surface contact angle of water could be increased from 27° up to 110°, and fluxes comparable to membranes commonly used for membrane distillation were achieved under similar process conditions. A 100 h test demonstrated the stability of the coating and the importance of using sufficiently stable base membranes.

Photoresponsive nanostructured membranes

KAUST Repository

Madhavan, Poornima; Sutisna, Burhannudin; Sougrat, Rachid; Nunes, Suzana Pereira

2016-01-01

The perspective of adding stimuli-response to isoporous membranes stimulates the development of separation devices with pores, which would open or close under control of environment chemical composition, temperature or exposure to light. Changes in pH and temperature have been previously investigated. In this work, we demonstrate for the first time the preparation of photoresponsive isoporous membranes, applying self-assembly non-solvent induced phase separation to a new light responsive block copolymer. First, we optimized the membrane formation by using poly(styrene-b-anthracene methyl methacrylate-b-methylmethacrylate) (PS-b-PAnMMA-b-PMMA) copolymer, identifying the most suitable solvent, copolymer block length, and other parameters. The obtained final triblock copolymer membrane morphologies were characterized using atomic force and electron microscopy. The microscopic analysis reveals that the PS-b-PAnMMA-b-PMMA copolymer can form both lamellar and ordered hexagonal nanoporous structures on the membrane top layer in appropriate solvent compositions. The nanostructured membrane emits fluorescence due to the presence of the anthracene mid-block. On irradiation of light the PS-b-PAnMMA-b-PMMA copolymer membranes has an additional stimuli response. The anthracene group undergoes conformational changes by forming [4 + 4] cycloadducts and this alters the membrane's water flux and solute retention. © 2016 The Royal Society of Chemistry.

Characteristics of membrane fouling in submerged membrane bioreactor under sub-critical flux operation.

Science.gov (United States)

Su, Y C; Huang, C P; Pan, Jill R; Lee, H C

2008-01-01

Recently, the membrane bioreactor (MBR) process has become one of the novel technologies to enhance the performance of biological treatment of wastewater. Membrane bioreactor process uses the membrane unit to replace a sediment tank, and this can greatly enhance treatment performance. However, membrane fouling in MBR restricts its widespread application because it leads to permeate flux decline, making more frequent membrane cleaning and replacement necessary, which then increases operating and maintenance costs. This study investigated the sludge characteristics in membrane fouling under sub-critical flux operation and also assessed the effect of shear stress on membrane fouling. Membrane fouling was slow under sub-critical flux operation. However, as filamentous microbes became dominant in the reactor, membrane fouling increased dramatically due to the increased viscosity and polysaccharides. A close link was found between membrane fouling and the amount of polysaccharides in soluble EPS. The predominant resistance was the cake resistance which could be minimized by increasing the shear stress. However, the resistance of colloids and solutes was not apparently reduced by increasing shear stress. Therefore, smaller particles such as macromolecules (e.g. polysaccharides) may play an important role in membrane fouling under sub-critical flux operation.

Autophagic kinases SmVPS34 and SmVPS15 are required for viability in the filamentous ascomycete Sordaria macrospora.

Science.gov (United States)

Voigt, Oliver; Herzog, Britta; Jakobshagen, Antonia; Pöggeler, Stefanie

2014-01-01

Autophagy is a tightly controlled degradation process of all eukaryotes. It includes the sequestration of cytoplasmic contents and organelles within a double-membraned autophagosome. Autophagy involves core autophagy related (atg) genes as well as genes regulating vesicle trafficking. Previously, we analyzed the impact of proteins of the core autophagic machinery SmATG7, SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. While deletion of Smatg8 and Smatg4 abolished fruiting-body formation and impaired vegetative growth, Smatg7 is required for viability. In yeast, the phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) and its myristoylated membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy and vacuolar protein sorting. However, their exact role in filamentous ascomycetes remains elusive. To determine the function of Smvps34 and Smvps15 we isolated genes with high sequence similarity to Saccharomyces cerevisiae VPS34 and VPS15. For both genes we were not able to generate a homokaryotic knockout mutant in S. macrospora, suggesting that Smvps34 and Smvps15 are required for viability. Furthermore, we analyzed the repertoire of vps genes encoded by S. macrospora and could identify putative homologs of nearly all of the 61 VPS genes of S. cerevisiae. Copyright © 2013 Elsevier GmbH. All rights reserved.

Emulsification using microporous membranes

Directory of Open Access Journals (Sweden)

Goran T. Vladisavljević

2011-10-01

Full Text Available Membrane emulsification is a process of injecting a pure dispersed phase or pre-emulsion through a microporous membrane into the continuous phase. As a result of the immiscibility of the two phases, droplets of the dispersed phase are formed at the outlets of membrane pores. The droplets formed in the process are removed from the membrane surface by applying cross-flow or stirring of the continuous phase or using a dynamic (rotating or vibrating membrane. The most commonly used membrane for emulsification is the Shirasu Porous Glass (SPG membrane, fabricated through spinodal decomposition in a melt consisting of Japanese volcanic ash (Shirasu, boric acid and calcium carbonate. Microsieve membranes are increasingly popular as an alternative to highly tortuous glass and ceramic membranes. Microsieves are usually fabricated from nickel by photolithography and electroplating or they can be manufactured from silicon nitride via Reactive Ion Etching (RIE. An advantage of microsieves compared to the SPG membrane is in much higher transmembrane fluxes and higher tolerance to fouling by the emulsion ingredients due to the existence of short, straight through pores. Unlike conventional emulsification devices such as high-pressure valve homogenisers and rotor-stator devices, membrane emulsification devices permit a precise control over the mean pore size over a wide range and during the process insignificant amount of energy is dissipated as heat. The drop size is primarily determined by the pore size, but it depends also on other parameters, such as membrane wettability, emulsion formulation, shear stress on the membrane surface, transmembrane pressure, etc.

Proton exchange membranes prepared by grafting of styrene/divinylbenzene into crosslinked PTFE membranes

International Nuclear Information System (INIS)

Li Jingye; Ichizuri, Shogo; Asano, Saneto; Mutou, Fumihiro; Ikeda, Shigetoshi; Iida, Minoru; Miura, Takaharu; Oshima, Akihiro; Tabata, Yoneho; Washio, Masakazu

2005-01-01

Thin PTFE membranes were prepared by coating the PTFE dispersion onto the aluminum films. Thus the thin crosslinked PTFE (RX-PTFE) membranes were obtained by means of electron beam irradiation above the melting temperature of PTFE under oxygen-free atmosphere. The RX-PTFE membranes were pre-irradiated and grafted by styrene with or without divinylbenzene (DVB) in liquid phase. The existence of DVB accelerated the initial grafting rate. The styrene grafted RX-PTFE membranes are white colored, on the other hand, the styrene/DVB grafted RX-PTFE membranes are colorless. The proton exchange membranes (PEMs) were obtained by sulfonating the grafted membranes using chlorosulfonic acid. The ion exchange capacity (IEC) values of the PEMs ranging from 1.5 to 2.8 meq/g were obtained. The PEMs made from the styrene/DVB grafted membranes showed higher chemical stability than those of the styrene grafted membranes under oxidative circumstance

Chorioamniotic membrane separation and preterm premature rupture of membranes complicating in utero myelomeningocele repair.

Science.gov (United States)

Soni, Shelly; Moldenhauer, Julie S; Spinner, Susan S; Rendon, Norma; Khalek, Nahla; Martinez-Poyer, Juan; Johnson, Mark P; Adzick, N Scott

2016-05-01

Since the results of the Management of Myelomeningocele Study were published, maternal-fetal surgery for the in utero treatment of spina bifida has become accepted as a standard of care alternative. Despite promise with fetal management of myelomeningocele repair, there are significant complications to consider. Chorioamniotic membrane separation and preterm premature rupture of membranes are known complications of invasive fetal procedures. Despite their relative frequency associated with fetal procedures, few data exist regarding risk factors that may be attributed to their occurrence or the natural history of pregnancies that are affected with chorionic membrane separation or preterm premature rupture of membranes related to the procedure. The objective of this study was to review chorioamniotic membrane separation and preterm premature rupture of membranes in a cohort of patients undergoing fetal management of myelomeningocele repair including identification of risk factors and outcomes. This was a retrospective review of patients undergoing fetal management of myelomeningocele repair and subsequent delivery from January 2011 through December 2013 at 1 institution. Patients were identified through the institutional fetal management of myelomeningocele repair database and chart review was performed. Perioperative factors and outcomes among patients with chorioamniotic membrane separation and preterm premature rupture of membranes were compared to those without. Risk factors associated with the development of chorioamniotic membrane separation and preterm premature rupture of membranes were determined. A total of 88 patients underwent fetal management of myelomeningocele repair and subsequently delivered during the study period. In all, 21 patients (23.9%) were diagnosed with chorioamniotic membrane separation by ultrasound and preterm premature rupture of membranes occurred in 27 (30.7%). Among the chorioamniotic membrane separation patients, 10 (47.6%) were

Pilot demonstration of energy-efficient membrane bioreactor (MBR) using reciprocating submerged membrane.

Science.gov (United States)

Ho, Jaeho; Smith, Shaleena; Patamasank, Jaren; Tontcheva, Petia; Kim, Gyu Dong; Roh, Hyung Keun

2015-03-01

Membrane bioreactor (MBR) is becoming popular for advanced wastewater treatment and water reuse. Air scouring to "shake" the membrane fibers is most suitable and applicable to maintain filtration without severe and rapidfouling. However, membrane fouling mitigating technologies are energy intensive. The goal of this research is to develop an alternative energy-saving MBR system to reduce energy consumption; a revolutionary system that will directly compete with air scouring technologies currently in the membrane water reuse market. The innovative MBR system, called reciprocation MBR (rMBR), prevents membrane fouling without the use of air scouring blowers. The mechanism featured is a mechanical reciprocating membrane frame that uses inertia to prevent fouling. Direct strong agitation of the fiber is also beneficial for the constant removal of solids built up on the membrane surface. The rMBR pilot consumes less energy than conventional coarse air scouring MBR systems. Specific energy consumption for membrane reciprocation for the pilot rMBR system was 0.072 kWh/m3 permeate produced at 40 LMH, which is 75% less than the conventional air scouring in an MBR system (0.29 kWh/m3). Reciprocation of the hollow-fiber membrane can overcome the hydrodynamic limitations of air scouring or cross-flow membrane systems with less energy consumption and/or higher energy efficiency.

Actin filaments growing against an elastic membrane: Effect of membrane tension

Science.gov (United States)

Sadhu, Raj Kumar; Chatterjee, Sakuntala

2018-03-01

We study the force generation by a set of parallel actin filaments growing against an elastic membrane. The elastic membrane tries to stay flat and any deformation from this flat state, either caused by thermal fluctuations or due to protrusive polymerization force exerted by the filaments, costs energy. We study two lattice models to describe the membrane dynamics. In one case, the energy cost is assumed to be proportional to the absolute magnitude of the height gradient (gradient model) and in the other case it is proportional to the square of the height gradient (Gaussian model). For the gradient model we find that the membrane velocity is a nonmonotonic function of the elastic constant μ and reaches a peak at μ =μ* . For μ membrane energy keeps increasing with time. For the Gaussian model, the system always reaches a steady state and the membrane velocity decreases monotonically with the elastic constant ν for all nonzero values of ν . Multiple filaments give rise to protrusions at different regions of the membrane and the elasticity of the membrane induces an effective attraction between the two protrusions in the Gaussian model which causes the protrusions to merge and a single wide protrusion is present in the system. In both the models, the relative time scale between the membrane and filament dynamics plays an important role in deciding whether the shape of elasticity-velocity curve is concave or convex. Our numerical simulations agree reasonably well with our analytical calculations.

Idiopathic epiretinal membrane

NARCIS (Netherlands)

Bu, Shao-Chong; Kuijer, Roelof; Li, Xiao-Rong; Hooymans, Johanna M M; Los, Leonoor I

2014-01-01

Background: Idiopathic epiretinal membrane (iERM) is a fibrocellular membrane that proliferates on the inner surface of the retina at the macular area. Membrane contraction is an important sight-threatening event and is due to fibrotic remodeling. Methods: Analysis of the current literature

Solid-state membrane module

Science.gov (United States)

Gordon, John Howard [Salt Lake City, UT; Taylor, Dale M [Murray, UT

2011-06-07

Solid-state membrane modules comprising at least one membrane unit, where the membrane unit has a dense mixed conducting oxide layer, and at least one conduit or manifold wherein the conduit or manifold comprises a dense layer and at least one of a porous layer and a slotted layer contiguous with the dense layer. The solid-state membrane modules may be used to carry out a variety of processes including the separating of any ionizable component from a feedstream wherein such ionizable component is capable of being transported through a dense mixed conducting oxide layer of the membrane units making up the membrane modules. For ease of construction, the membrane units may be planar.

Vacuolar H+ -ATPase c protects glial cell death induced by sodium nitroprusside under glutathione-depleted condition.

Science.gov (United States)

Byun, Yu Jeong; Lee, Seong-Beom; Lee, Hwa Ok; Son, Min Jeong; Kim, Ho-Shik; Kwon, Oh-Joo; Jeong, Seong-Whan

2011-08-01

We examined the role of the c subunit (ATP6L) of vacuolar H(+) -ATPase and its molecular mechanisms in glial cell death induced by sodium nitroprusside (SNP). ATP6L siRNA-transfected cells treated with SNP showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, but reduction of ATP6L did not affect the regulation of lysosomal pH in analyses with lysosomal pH-dependent fluorescence probes. Photodegraded SNP and ferrous sulfate induced cytotoxicity with the same pattern as that of SNP, but SNAP and potassium cyanide did not show activity. Pretreatment of the transfected cells with deferoxamine (DFO) reduced ROS production and significantly inhibited the cytotoxicity, which indicates that primarily iron rather than nitric oxide or cyanide from SNP contributes to cell death. Involvement of apoptotic processes in the cells was not shown. Pretreatment with JNK or p38 chemical inhibitor significantly inhibited the cytotoxicity, and we also confirmed that the MAPKs were activated in the cells by immunoblot analysis. Significant increase of LC3-II conversion was observed in the cells, and the conversions were inhibited by cotransfection of the MAPK siRNAs and pretreatment with DFO. Introduction of Atg5 siRNA inhibited the cytotoxicity and inhibited the activation of MAPKs and the conversion of LC3. We finally confirmed autophagic cell death and involvement of MAPKs by observation of autophagic vacuoles via electron microscopy. These data suggest that ATP6L has a protective role against SNP-induced autophagic cell death via inhibition of JNK and p38 in GSH-depleted glial cells. Copyright © 2011 Wiley-Liss, Inc.

Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9.

Science.gov (United States)

Zhang, Jingbo; Baetz, Ulrike; Krügel, Undine; Martinoia, Enrico; De Angeli, Alexis

2013-10-01

Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.

The Role of Ion Exchange Membranes in Membrane Capacitive Deionisation.

Science.gov (United States)

Hassanvand, Armineh; Wei, Kajia; Talebi, Sahar; Chen, George Q; Kentish, Sandra E

2017-09-14

Ion-exchange membranes (IEMs) are unique in combining the electrochemical properties of ion exchange resins and the permeability of a membrane. They are being used widely to treat industrial effluents, and in seawater and brackish water desalination. Membrane Capacitive Deionisation (MCDI) is an emerging, energy efficient technology for brackish water desalination in which these ion-exchange membranes act as selective gates allowing the transport of counter-ions toward carbon electrodes. This article provides a summary of recent developments in the preparation, characterization, and performance of ion exchange membranes in the MCDI field. In some parts of this review, the most relevant literature in the area of electrodialysis (ED) is also discussed to better elucidate the role of the ion exchange membranes. We conclude that more work is required to better define the desalination performance of the proposed novel materials and cell designs for MCDI in treating a wide range of feed waters. The extent of fouling, the development of cleaning strategies, and further techno-economic studies, will add value to this emerging technique.

Model cell membranes

DEFF Research Database (Denmark)

Günther-Pomorski, Thomas; Nylander, Tommy; Cardenas Gomez, Marite

2014-01-01

The high complexity of biological membranes has motivated the development and application of a wide range of model membrane systems to study biochemical and biophysical aspects of membranes in situ under well defined conditions. The aim is to provide fundamental understanding of processes control...

The secret life of tethers: the role of tethering factors in SNARE complex regulation

Directory of Open Access Journals (Sweden)

Michelle L Dubuke

2016-05-01

Full Text Available Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the interplay of the multisubunit tethering complexes (MTC with the SNAREs and Sec1/Munc18 (SM proteins. Although these factors are used in different stages of membrane trafficking, e.g. Golgi to plasma membrane transport vs vacuolar fusion, and in a variety of diverse eukaryotic cell types, many commonalities between their functions are being revealed. We explore the various protein-protein interactions and findings from functional reconstitution studies in order to highlight both their common features and the differences in their modes of regulation. These studies serve as a starting point for mechanistic explorations in other systems.

How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

Science.gov (United States)

Golubovic, Leonardo; Gao, Lianghui; Chen, Licui; Fang, Weihai

2012-02-01

In this study, coarse grained Dissipative Particle Dynamics simulation with implementation of electrostatic interactions is developed in constant pressure and surface tension ensemble to elucidate how the antimicrobial peptide molecules affect bilayer cell membrane structure and kill bacteria. We find that peptides with different chemical-physical properties exhibit different membrane obstructing mechanisms. Peptide molecules can destroy vital functions of the affected bacteria by translocating across their membranes via worm-holes, or by associating with membrane lipids to form hydrophilic cores trapped inside the hydrophobic domain of the membranes. In the latter scenario, the affected membranes are strongly corrugated (buckled) in accord with very recent experimental observations [G. E. Fantner et al., Nat. Nanotech., 5 (2010), pp. 280-285].

Catalytic nanoporous membranes

Science.gov (United States)

Pellin, Michael J; Hryn, John N; Elam, Jeffrey W

2013-08-27

A nanoporous catalytic membrane which displays several unique features Including pores which can go through the entire thickness of the membrane. The membrane has a higher catalytic and product selectivity than conventional catalysts. Anodic aluminum oxide (AAO) membranes serve as the catalyst substrate. This substrate is then subjected to Atomic Layer Deposition (ALD), which allows the controlled narrowing of the pores from 40 nm to 10 nm in the substrate by deposition of a preparatory material. Subsequent deposition of a catalytic layer on the inner surfaces of the pores reduces pore sizes to less than 10 nm and allows for a higher degree of reaction selectivity. The small pore sizes allow control over which molecules enter the pores, and the flow-through feature can allow for partial oxidation of reactant species as opposed to complete oxidation. A nanoporous separation membrane, produced by ALD is also provided for use in gaseous and liquid separations. The membrane has a high flow rate of material with 100% selectivity. Also provided is a method for producing a catalytic membrane having flow-through pores and discreet catalytic clusters adhering to the inside surfaces of the pores.

Sweeping Gas Membrane Desalination Using Commercial Hydrophobic Hollow Fiber Membranes; TOPICAL

International Nuclear Information System (INIS)

EVANS, LINDSEY; MILLER, JAMES E.

2002-01-01

Water shortages affect 88 developing countries that are home to half of the world's population. In these places, 80-90% of all diseases and 30% of all deaths result from poor water quality. Furthermore, over the next 25 years, the number of people affected by severe water shortages is expected to increase fourfold. Low cost methods of purifying freshwater, and desalting seawater are required to contend with this destabilizing trend. Membrane distillation (MD) is an emerging technology for separations that are traditionally accomplished via conventional distillation or reverse osmosis. As applied to desalination, MD involves the transport of water vapor from a saline solution through the pores of a hydrophobic membrane. In sweeping gas MD, a flowing gas stream is used to flush the water vapor from the permeate side of the membrane, thereby maintaining the vapor pressure gradient necessary for mass transfer. Since liquid does not penetrate the hydrophobic membrane, dissolved ions are completely rejected by the membrane. MD has a number of potential advantages over conventional desalination including low temperature and pressure operation, reduced membrane strength requirements, compact size, and 100% rejection of non-volatiles. The present work evaluated the suitability of commercially available technology for sweeping gas membrane desalination. Evaluations were conducted with Celgard Liqui-Cel(reg s ign) Extra-Flow 2.5X8 membrane contactors with X-30 and X-40 hydrophobic hollow fiber membranes. Our results show that sweeping gas membrane desalination systems are capable of producing low total dissolved solids (TDS) water, typically 10 ppm or less, from seawater, using low grade heat. However, there are several barriers that currently prevent sweeping gas MD from being a viable desalination technology. The primary problem is that large air flows are required to achieve significant water yields, and the costs associated with transporting this air are prohibitive. To

Polymalic Acid Tritryptophan Copolymer Interacts with Lipid Membrane Resulting in Membrane Solubilization

Directory of Open Access Journals (Sweden)

Hui Ding

2017-01-01

Full Text Available Anionic polymers with membrane permeation functionalities are highly desirable for secure cytoplasmic drug delivery. We have developed tritryptophan containing copolymer (P/WWW of polymalic acid (PMLA that permeates membranes by a mechanism different from previously described PMLA copolymers of trileucine (P/LLL and leucine ethyl ester (P/LOEt that use the “barrel stave” and “carpet” mechanism, respectively. The novel mechanism leads to solubilization of membranes by forming copolymer “belts” around planar membrane “packages.” The formation of such packages is supported by results obtained from studies including size-exclusion chromatography, confocal microscopy, and fluorescence energy transfer. According to this “belt” mechanism, it is hypothesized that P/WWW first attaches to the membrane surface. Subsequently the hydrophobic tryptophan side chains translocate into the periphery and insert into the lipid bilayer thereby cutting the membrane into packages. The reaction is driven by the high affinity between the tryptophan residues and lipid side chains resulting in a stable configuration. The formation of the membrane packages requires physical agitation suggesting that the success of the translocation depends on the fluidity of the membrane. It is emphasized that the “belt” mechanism could specifically function in the recognition of abnormal cells with high membrane fluidity and in response to hyperthermia.

Membrane Distillation of Meat Industry Effluent with Hydrophilic Polyurethane Coated Polytetrafluoroethylene Membranes

Directory of Open Access Journals (Sweden)

M. G. Mostafa

2017-09-01

Full Text Available Meat rendering operations produce stick water waste which is rich in proteins, fats, and minerals. Membrane distillation (MD may further recover water and valuable solids, but hydrophobic membranes are contaminated by the fats. Here, commercial hydrophobic polytetrafluorethylene (PTFE membranes with a hydrophilic polyurethane surface layer (PU-PTFE are used for the first time for direct contact MD (DCMD on real poultry, fish, and bovine stick waters. Metal membrane microfiltration (MMF was also used to capture fats prior to MD. Although the standard hydrophobic PTFE membranes failed rapidly, PU-PTFE membranes effectively processed all stick water samples to colourless permeate with sodium rejections >99%. Initial clean solution fluxes 5–6 L/m2/h declined to less than half during short 40% water recovery tests for all stick water samples. Fish stick water uniquely showed reduced fouling and up to 78% water recovery. Lost flux was easily restored by rinsing the membrane with clean water. MMF prior to MD removed 92% of fats, facilitating superior MD performance. Differences in fouling between stick waters were attributed to temperature polarisation from higher melt temperature fats and relative proportions to proteins. Hydrophilic coated MD membranes are applicable to stick water processing but further studies should consider membrane cleaning and longer-term stability.

Membrane Distillation of Meat Industry Effluent with Hydrophilic Polyurethane Coated Polytetrafluoroethylene Membranes.

Science.gov (United States)

Mostafa, M G; Zhu, Bo; Cran, Marlene; Dow, Noel; Milne, Nicholas; Desai, Dilip; Duke, Mikel

2017-09-29

Meat rendering operations produce stick water waste which is rich in proteins, fats, and minerals. Membrane distillation (MD) may further recover water and valuable solids, but hydrophobic membranes are contaminated by the fats. Here, commercial hydrophobic polytetrafluorethylene (PTFE) membranes with a hydrophilic polyurethane surface layer (PU-PTFE) are used for the first time for direct contact MD (DCMD) on real poultry, fish, and bovine stick waters. Metal membrane microfiltration (MMF) was also used to capture fats prior to MD. Although the standard hydrophobic PTFE membranes failed rapidly, PU-PTFE membranes effectively processed all stick water samples to colourless permeate with sodium rejections >99%. Initial clean solution fluxes 5-6 L/m²/h declined to less than half during short 40% water recovery tests for all stick water samples. Fish stick water uniquely showed reduced fouling and up to 78% water recovery. Lost flux was easily restored by rinsing the membrane with clean water. MMF prior to MD removed 92% of fats, facilitating superior MD performance. Differences in fouling between stick waters were attributed to temperature polarisation from higher melt temperature fats and relative proportions to proteins. Hydrophilic coated MD membranes are applicable to stick water processing but further studies should consider membrane cleaning and longer-term stability.

Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

Science.gov (United States)

Fabrizio, Paola; Hoon, Shawn; Shamalnasab, Mehrnaz; Galbani, Abdulaye; Wei, Min; Giaever, Guri; Nislow, Corey; Longo, Valter D

2010-07-15

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Magnetically controlled permeability membranes

KAUST Repository

Kosel, Jurgen

2013-10-31

A bioactive material delivery system can include a thermoresponsive polymer membrane and nanowires distributed within the thermoresponsive polymer membrane. Magnetic activation of a thermoresponsive polymer membrane can take place via altering the magnetization or dimensions of nanowires dispersed or ordered within the membrane matrix.

Magnetically controlled permeability membranes

KAUST Repository

Kosel, Jü rgen; Khashab, Niveen M.; Zaher, Amir

2013-01-01

A bioactive material delivery system can include a thermoresponsive polymer membrane and nanowires distributed within the thermoresponsive polymer membrane. Magnetic activation of a thermoresponsive polymer membrane can take place via altering the magnetization or dimensions of nanowires dispersed or ordered within the membrane matrix.

Chapter 6: cubic membranes the missing dimension of cell membrane organization.

Science.gov (United States)

Almsherqi, Zakaria A; Landh, Tomas; Kohlwein, Sepp D; Deng, Yuru

2009-01-01

Biological membranes are among the most fascinating assemblies of biomolecules: a bilayer less than 10 nm thick, composed of rather small lipid molecules that are held together simply by noncovalent forces, defines the cell and discriminates between "inside" and "outside", survival, and death. Intracellular compartmentalization-governed by biomembranes as well-is a characteristic feature of eukaryotic cells, which allows them to fulfill multiple and highly specialized anabolic and catabolic functions in strictly controlled environments. Although cellular membranes are generally visualized as flat sheets or closely folded isolated objects, multiple observations also demonstrate that membranes may fold into "unusual", highly organized structures with 2D or 3D periodicity. The obvious correlation of highly convoluted membrane organizations with pathological cellular states, for example, as a consequence of viral infection, deserves close consideration. However, knowledge about formation and function of these highly organized 3D periodic membrane structures is scarce, primarily due to the lack of appropriate techniques for their analysis in vivo. Currently, the only direct way to characterize cellular membrane architecture is by transmission electron microscopy (TEM). However, deciphering the spatial architecture solely based on two-dimensionally projected TEM images is a challenging task and prone to artifacts. In this review, we will provide an update on the current progress in identifying and analyzing 3D membrane architectures in biological systems, with a special focus on membranes with cubic symmetry, and their potential role in physiological and pathophysiological conditions. Proteomics and lipidomics approaches in defined experimental cell systems may prove instrumental to understand formation and function of 3D membrane morphologies.

Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases

DEFF Research Database (Denmark)

Larsen, Jannik Bruun; Jensen, Martin Borch; Bhatia, Vikram Kjøller

2015-01-01

Trafficking and sorting of membrane-anchored Ras GTPases are regulated by partitioning between distinct membrane domains. Here, in vitro experiments and microscopic molecular theory reveal membrane curvature as a new modulator of N-Ras lipid anchor and palmitoyl chain partitioning. Membrane...

Membrane properties for permeability testing: Skin versus synthetic membranes.

Science.gov (United States)

Haq, Anika; Dorrani, Mania; Goodyear, Benjamin; Joshi, Vivek; Michniak-Kohn, Bozena

2018-03-25

Synthetic membranes that are utilized in diffusion studies for topical and transdermal formulations are usually porous thin polymeric sheets for example cellulose acetate (CA) and polysulfones. In this study, the permeability of human skin was compared using two synthetic membranes: cellulose acetate and Strat-M® membrane and lipophilic and hydrophilic compounds either as saturated or formulated solutions as well as marketed dosage forms. Our data suggests that hydrophilic compounds have higher permeation in Strat-M membranes compared with lipophilic ones. High variation in permeability values, a typical property of biological membranes, was not observed with Strat-M. In addition, the permeability of Strat-M was closer to that of human skin than that of cellulose acetate (CA > Strat-M > Human skin). Our results suggest that Strat-M with little or no lot to lot variability can be applied in pilot studies of diffusion tests instead of human skin and is a better substitute than a cellulose acetate. Copyright © 2018 Elsevier B.V. All rights reserved.

Probing lipid membrane electrostatics

Science.gov (United States)

Yang, Yi

The electrostatic properties of lipid bilayer membranes play a significant role in many biological processes. Atomic force microscopy (AFM) is highly sensitive to membrane surface potential in electrolyte solutions. With fully characterized probe tips, AFM can perform quantitative electrostatic analysis of lipid membranes. Electrostatic interactions between Silicon nitride probes and supported zwitterionic dioleoylphosphatidylcholine (DOPC) bilayer with a variable fraction of anionic dioleoylphosphatidylserine (DOPS) were measured by AFM. Classical Gouy-Chapman theory was used to model the membrane electrostatics. The nonlinear Poisson-Boltzmann equation was numerically solved with finite element method to provide the potential distribution around the AFM tips. Theoretical tip-sample electrostatic interactions were calculated with the surface integral of both Maxwell and osmotic stress tensors on tip surface. The measured forces were interpreted with theoretical forces and the resulting surface charge densities of the membrane surfaces were in quantitative agreement with the Gouy-Chapman-Stern model of membrane charge regulation. It was demonstrated that the AFM can quantitatively detect membrane surface potential at a separation of several screening lengths, and that the AFM probe only perturbs the membrane surface potential by external field created by the internai membrane dipole moment. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported DOPC membranes. This new ability to quantitatively measure the membrane dipole density in a noninvasive manner will be useful in identifying the biological effects of the dipole potential. Finally, heterogeneous model membranes were studied with fluid electric force microscopy (FEFM). Electrostatic mapping was demonstrated with 50 nm resolution. The capabilities of quantitative electrostatic measurement and lateral charge density mapping make AFM a unique and powerful

Application of the nanocomposite membrane as electrolyte of proton exchange membrane fuel cell

International Nuclear Information System (INIS)

Mahreni

2010-01-01

Hydrogen fuel cells proton exchange membrane fuel cell (PEMFC) is currently still in development and commercialization. Several barriers to the commercialization of these Nafion membrane as electrolyte is its very sensitive to humidity fluctuation. Nafion must be modified by making a composite Nafion-SiO 2 -HPA to increase electrolyte resistance against humidity fluctuations during the cell used. Research carried out by mixing Nafion solution with Tetra Ethoxy Ortho Silicate (TEOS) and conductive materials is phosphotungstic acid (PWA) by varying the ratio of Nafion, TEOS and PWA. The membrane is produced by heating a mixture of Nafion, TEOS and PWA by varying the evaporation temperature, time and annealing temperature to obtain the transparent membrane. The resulting membrane was analyzed its physical, chemical and electrochemical properties by applying the membrane as electrolyte of PEMFC at various humidity and temperature of operation. The results showed that at low temperatures (30-90 °C) and high humidity at 100 % RH, pure Nafion membrane is better than composite membrane (Nafion-SiO 2 -PWA), but at low humidity condition composite membrane is better than the pure Nafion membrane. It can be concluded that the composite membranes of (Nafion-SiO 2 -PWA) can be used as electrolyte of PEMFC operated at low humidity (40 % RH) and temperature between (30-90 °C). (author)

Radiation-induced damage of membranes

International Nuclear Information System (INIS)

Yonei, Shuji

1977-01-01

An outline of membranous structure was stated, and radiation-induced damage of membranes were surveyed. By irradiation, permeability of membranes, especially passive transportation mechanism, was damaged, and glycoprotein in the surface layers of cells and the surface layer structures were changed. The intramembranous damage was induced by decrease of electrophoresis of nuclear mambranes and a quantitative change of cytochrome P450 of microsomal membranes of the liver, and peroxidation of membranous lipid and SH substitute damage of membranous protein were mentioned as the mechanism of membranous damage. Recovery of membranous damage depends on radiation dose and temperature, and membranous damage participates largely in proliferation death. (tsunoda, M.)

Studies on membrane for redox flow battery. 9. Crosslinking of the membrane by the electron radiation and durability of the membrane

International Nuclear Information System (INIS)

Ohya, Haruhiko; Minamihira, Kazunori; Hwang, Gab-Jin; Kawahara, Takashi; Aihara, Masahiko; Negishi, Youichi; Kang, An-Soo.

1995-01-01

Chlorosulfonated homogeneous and asymmetric cation exchange membranes were tested as separators for the all-vanadium redox flow battery. The membrane was prepared by chlorosulfonation of the polyethylene film in vapour phase. In the case of the polyethylene film of 20 μm thickness used for the homogeneous membrane, area resistivity of 0.5 Ω · cm 2 in 2M KCl aq. solution was reached at 120 min. chlorosulfonation time. In the case of heat laminated 20 μm thick PE film on a neutral porous polyolefin film of 200 μm thickness used for the asymmetric membrane, a minimum area resistivity of 1 Ω · cm 2 in 2M KCl was achieved at 120 min. chlorosulfonation time. The performance evaluation of the membranes as separators in the all-vanadium redox flow battery was also measured. The area resistivity of the membranes in the measuring-cell using charge-discharge current density 63.7 mA/cm 2 was 1.4 Ω · cm 2 and 2.2 Ω · cm 2 for charge and discharge respectively for the homogeneous membrane, and 3.6 Ω · cm 2 and 4.3 Ω · cm 2 for charge discharge cycles respectively for the asymmetric membrane. The chlorosulfonated homogeneous cation exchange membrane was cross-linked by the electron radiation to improve durability of the membrane. The crosslinked membrane which has the high degree of cross-linking, did not shown the mechanical breakage by swelling or shrinking in the acidic vanadium solution, but its area resistivity in the all-vanadium redox flow battery was increased. (author)

Primordial membranes

DEFF Research Database (Denmark)

Hanczyc, Martin M; Monnard, Pierre-Alain

2017-01-01

Cellular membranes, which are self-assembled bilayer structures mainly composed of lipids, proteins and conjugated polysaccharides, are the defining feature of cell physiology. It is likely that the complexity of contemporary cells was preceded by simpler chemical systems or protocells during...... the various evolutionary stages that led from inanimate to living matter. It is also likely that primitive membranes played a similar role in protocell 'physiology'. The composition of such ancestral membranes has been proposed as mixtures of single hydrocarbon chain amphiphiles, which are simpler versions...

Recovery of real dye bath wastewater using integrated membrane process: considering water recovery, membrane fouling and reuse potential of membranes.

Science.gov (United States)

Balcik-Canbolat, Cigdem; Sengezer, Cisel; Sakar, Hacer; Karagunduz, Ahmet; Keskinler, Bulent

2017-11-01

It has been recognized by the whole world that textile industry which produce large amounts of wastewater with strong color and toxic organic compounds is a major problematical industry requiring effective treatment solutions. In this study, reverse osmosis (RO) membranes were tested on biologically treated real dye bath wastewater with and without pretreatment by nanofiltration (NF) membrane to recovery. Also membrane fouling and reuse potential of membranes were investigated by multiple filtrations. Obtained results showed that only NF is not suitable to produce enough quality to reuse the wastewater in a textile industry as process water while RO provide successfully enough permeate quality. The results recommend that integrated NF/RO membrane process is able to reduce membrane fouling and allow long-term operation for real dye bath wastewater.

Vacuum membrane distillation of liquid desiccants Utilizing Hollow Fiber Membranes

KAUST Repository

Lefers, Ryan

2018-01-31

This paper documents the testing of a vacuum membrane distillation system intended for use with liquid desiccants. Liquid desiccants offer the possibility for low-energy, ambient temperature dehumidification. Effective desalination and purification of diluted desiccants outputs two important products: a concentrated desiccant for reuse in dehumidification and fresh water. In this study, vacuum membrane distillation was used in the laboratory to purify diluted liquid desiccants. Calcium chloride and magnesium chloride were the desiccants selected for testing. Desiccant solutions were pumped through the lumens of poly(vinylidene fluoride) (PVDF) hollow fiber membranes at varying feed inlet temperatures, solution velocity rates and vacuum set points during membrane distillation. An average flux of 8 kg m-2 h-1 was obtained using 30 wt% magnesium chloride solution at a temperature of 50 °C while applying vacuum to achieve 25 mbar absolute pressure on the air side of the membrane. The results are promising for the development of a full-scale vacuum membrane distillation process for desiccant solution regeneration and fresh water recovery. In addition, the recovered condensate was of sufficient quality for use in agricultural irrigation or drinking water.

Vacuum membrane distillation of liquid desiccants Utilizing Hollow Fiber Membranes

KAUST Repository

Lefers, Ryan; Srivatsa Bettahalli, N.M.; Fedoroff, Nina V.; Nunes, Suzana Pereira; Leiknes, TorOve

2018-01-01

This paper documents the testing of a vacuum membrane distillation system intended for use with liquid desiccants. Liquid desiccants offer the possibility for low-energy, ambient temperature dehumidification. Effective desalination and purification of diluted desiccants outputs two important products: a concentrated desiccant for reuse in dehumidification and fresh water. In this study, vacuum membrane distillation was used in the laboratory to purify diluted liquid desiccants. Calcium chloride and magnesium chloride were the desiccants selected for testing. Desiccant solutions were pumped through the lumens of poly(vinylidene fluoride) (PVDF) hollow fiber membranes at varying feed inlet temperatures, solution velocity rates and vacuum set points during membrane distillation. An average flux of 8 kg m-2 h-1 was obtained using 30 wt% magnesium chloride solution at a temperature of 50 °C while applying vacuum to achieve 25 mbar absolute pressure on the air side of the membrane. The results are promising for the development of a full-scale vacuum membrane distillation process for desiccant solution regeneration and fresh water recovery. In addition, the recovered condensate was of sufficient quality for use in agricultural irrigation or drinking water.

Development of composite membranes of PVA-TEOS doped KOH for alkaline membrane fuel cell

International Nuclear Information System (INIS)

Haryadi,; Sugianto, D.; Ristopan, E.

2015-01-01

Anion exchange membranes (AEMs) play an important role in separating fuel and oxygen (or air) in the Alkaline Membrane Fuel Cells. Preparation of hybrid organic inorganic materials of Polyvinylalcohol (PVA) - Tetraethylorthosilicate (TEOS) composite membrane doped KOH for direct alcohol alkaline fuel cell application has been investigated. The sol-gel method has been used to prepare the composite membrane of PVA-TEOS through crosslinking step and catalyzed by concentrated of hydrochloric acid. The gel solution was cast on the membrane plastic plate to obtain membrane sheets. The dry membranes were then doped by immersing in various concentrations of KOH solutions for about 4 hours. Investigations of the cross-linking process and the presence of hydroxyl group were conducted by FTIR as shown for frequency at about 1600 cm −1 and 3300 cm −1 respectively. The degree of swelling in ethanol decreased as the KOH concentration for membrane soaking process increased. The ion exchange capacity (IEC) of the membrane was 0.25meq/g. This composite membranes display significant ionic conductivity of 3.23 x 10 −2 S/cm in deionized water at room temperature. In addition, the morphology observation by scanning electron microscope (SEM) of the membrane indicates that soaking process of membrane in KOH increased thermal resistant

Development of composite membranes of PVA-TEOS doped KOH for alkaline membrane fuel cell

Science.gov (United States)

Haryadi, Sugianto, D.; Ristopan, E.

2015-12-01

Anion exchange membranes (AEMs) play an important role in separating fuel and oxygen (or air) in the Alkaline Membrane Fuel Cells. Preparation of hybrid organic inorganic materials of Polyvinylalcohol (PVA) - Tetraethylorthosilicate (TEOS) composite membrane doped KOH for direct alcohol alkaline fuel cell application has been investigated. The sol-gel method has been used to prepare the composite membrane of PVA-TEOS through crosslinking step and catalyzed by concentrated of hydrochloric acid. The gel solution was cast on the membrane plastic plate to obtain membrane sheets. The dry membranes were then doped by immersing in various concentrations of KOH solutions for about 4 hours. Investigations of the cross-linking process and the presence of hydroxyl group were conducted by FTIR as shown for frequency at about 1600 cm-1 and 3300 cm-1 respectively. The degree of swelling in ethanol decreased as the KOH concentration for membrane soaking process increased. The ion exchange capacity (IEC) of the membrane was 0.25meq/g. This composite membranes display significant ionic conductivity of 3.23 x 10-2 S/cm in deionized water at room temperature. In addition, the morphology observation by scanning electron microscope (SEM) of the membrane indicates that soaking process of membrane in KOH increased thermal resistant.

Development of composite membranes of PVA-TEOS doped KOH for alkaline membrane fuel cell

Energy Technology Data Exchange (ETDEWEB)

Haryadi,, E-mail: haryadi@polban.ac.id; Sugianto, D.; Ristopan, E. [Department of Chemical Engineering, Politeknik Negeri Bandung Jl. Gegerkalong Hilir, Ds. Ciwaruga, Bandung West Java (Indonesia)

2015-12-29

Anion exchange membranes (AEMs) play an important role in separating fuel and oxygen (or air) in the Alkaline Membrane Fuel Cells. Preparation of hybrid organic inorganic materials of Polyvinylalcohol (PVA) - Tetraethylorthosilicate (TEOS) composite membrane doped KOH for direct alcohol alkaline fuel cell application has been investigated. The sol-gel method has been used to prepare the composite membrane of PVA-TEOS through crosslinking step and catalyzed by concentrated of hydrochloric acid. The gel solution was cast on the membrane plastic plate to obtain membrane sheets. The dry membranes were then doped by immersing in various concentrations of KOH solutions for about 4 hours. Investigations of the cross-linking process and the presence of hydroxyl group were conducted by FTIR as shown for frequency at about 1600 cm{sup −1} and 3300 cm{sup −1} respectively. The degree of swelling in ethanol decreased as the KOH concentration for membrane soaking process increased. The ion exchange capacity (IEC) of the membrane was 0.25meq/g. This composite membranes display significant ionic conductivity of 3.23 x 10{sup −2} S/cm in deionized water at room temperature. In addition, the morphology observation by scanning electron microscope (SEM) of the membrane indicates that soaking process of membrane in KOH increased thermal resistant.

Fabrication of electrospun nanofibrous membranes for membrane distillation application

KAUST Repository

Francis, Lijo; Maab, Husnul; Alsaadi, Ahmad Salem; Nunes, Suzana Pereira; Ghaffour, NorEddine; Amy, Gary L.

2013-01-01

Nanofibrous membranes of Matrimid have been successfully fabricated using an electrospinning technique under optimized conditions. Nanofibrous membranes are found to be highly hydrophobic with a high water contact angle of 130°. Field emission

Effect of dope solution temperature on the membrane structure and membrane distillation performance

Science.gov (United States)

Nawi, N. I. M.; Bilad, M. R.; Nordin, N. A. H. M.

2018-04-01

Membrane distillation (MD) is a non-isothermal process applicable to purify water using hydrophobic membrane. Membrane in MD is hydrophobic, permeable to water vapor but repels liquid water. MD membrane is expected to pose high flux, high fouling and scaling resistances and most importantly high wetting resistance. This study develops flat-sheet polyvinylidene fluoride (PVDF) membrane by exploring both liquid-liquid and liquid-solid phase inversion technique largely to improve its wetting resistance and flux performance. We hypothesize that temperature of dope solution play roles in solid-liquid separation during membrane formation and an optimum balance between liquid-liquid and liquid-solid (crystallization) separation leads to highly performance PVDF membrane. Findings obtained from differential scanning calorimeter test show that increasing dope solution temperature reduces degree of PVDF crystallinity and suppresses formation of crystalline structure. The morphological images of the resulting membranes show that at elevated dope solution temperature (40, 60, 80 and 100°C), the spherulite-like structures are formed across the thickness of membranes ascribed from due to different type of crystals. The performance of direct-contact MD shows that the obtained flux of the optimum dope temperature (60°C) of 10.8 L/m2h is comparable to commercial PTFE-based MD membrane.

Anion exchange membrane

Science.gov (United States)

Verkade, John G; Wadhwa, Kuldeep; Kong, Xueqian; Schmidt-Rohr, Klaus

2013-05-07

An anion exchange membrane and fuel cell incorporating the anion exchange membrane are detailed in which proazaphosphatrane and azaphosphatrane cations are covalently bonded to a sulfonated fluoropolymer support along with anionic counterions. A positive charge is dispersed in the aforementioned cations which are buried in the support to reduce the cation-anion interactions and increase the mobility of hydroxide ions, for example, across the membrane. The anion exchange membrane has the ability to operate at high temperatures and in highly alkaline environments with high conductivity and low resistance.

Specific ion effects on membrane potential and the permselectivity of ion exchange membranes.

Science.gov (United States)

Geise, Geoffrey M; Cassady, Harrison J; Paul, Donald R; Logan, Bruce E; Hickner, Michael A

2014-10-21

Membrane potential and permselectivity are critical parameters for a variety of electrochemically-driven separation and energy technologies. An electric potential is developed when a membrane separates electrolyte solutions of different concentrations, and a permselective membrane allows specific species to be transported while restricting the passage of other species. Ion exchange membranes are commonly used in applications that require advanced ionic electrolytes and span technologies such as alkaline batteries to ammonium bicarbonate reverse electrodialysis, but membranes are often only characterized in sodium chloride solutions. Our goal in this work was to better understand membrane behaviour in aqueous ammonium bicarbonate, which is of interest for closed-loop energy generation processes. Here we characterized the permselectivity of four commercial ion exchange membranes in aqueous solutions of sodium chloride, ammonium chloride, sodium bicarbonate, and ammonium bicarbonate. This stepwise approach, using four different ions in aqueous solution, was used to better understand how these specific ions affect ion transport in ion exchange membranes. Characterization of cation and anion exchange membrane permselectivity, using these ions, is discussed from the perspective of the difference in the physical chemistry of the hydrated ions, along with an accompanying re-derivation and examination of the basic equations that describe membrane potential. In general, permselectivity was highest in sodium chloride and lowest in ammonium bicarbonate solutions, and the nature of both the counter- and co-ions appeared to influence measured permselectivity. The counter-ion type influences the binding affinity between counter-ions and polymer fixed charge groups, and higher binding affinity between fixed charge sites and counter-ions within the membrane decreases the effective membrane charge density. As a result permselectivity decreases. The charge density and polarizability

3D pressure field in lipid membranes and membrane-protein complexes

DEFF Research Database (Denmark)

Ollila, O H Samuli; Risselada, H Jelger; Louhivuori, Martti

2009-01-01

We calculate full 3D pressure fields for inhomogeneous nanoscale systems using molecular dynamics simulation data. The fields represent systems with increasing level of complexity, ranging from semivesicles and vesicles to membranes characterized by coexistence of two phases, including also...... a protein-membrane complex. We show that the 3D pressure field is distinctly different for curved and planar bilayers, the pressure field depends strongly on the phase of the membrane, and that an integral protein modulates the tension and elastic properties of the membrane....

Improving Hemocompatibility of Membranes for Extracorporeal Membrane Oxygenators by Grafting Nonthrombogenic Polymer Brushes.

Science.gov (United States)

Obstals, Fabian; Vorobii, Mariia; Riedel, Tomáš; de Los Santos Pereira, Andres; Bruns, Michael; Singh, Smriti; Rodriguez-Emmenegger, Cesar

2018-03-01

Nonthrombogenic modifications of membranes for extracorporeal membrane oxygenators (ECMOs) are of key interest. The absence of hemocompatibility of these membranes and the need of anticoagulation of patients result in severe and potentially life-threatening complications during ECMO treatment. To address the lack of hemocompatibility of the membrane, surface modifications are developed, which act as barriers to protein adsorption on the membrane and, in this way, prevent activation of the coagulation cascade. The modifications are based on nonionic and zwitterionic polymer brushes grafted directly from poly(4-methyl-1-pentene) (TPX) membranes via single electron transfer-living radical polymerization. Notably, this work introduces the first example of well-controlled surface-initiated radical polymerization of zwitterionic brushes. The antifouling layers markedly increase the recalcification time (a proxy of initiation of coagulation) compared to bare TPX membranes. Furthermore, platelet and leukocyte adhesion is drastically decreased, rendering the ECMO membranes hemocompatible. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improved surface property of PVDF membrane with amphiphilic zwitterionic copolymer as membrane additive

Energy Technology Data Exchange (ETDEWEB)

Li Jianhua, E-mail: jhli_2005@163.com [Institute of Biomedical and Pharmaceutical Technology and College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350001 (China); Li Mizi; Miao Jing; Wang Jiabin; Shao Xisheng [Institute of Biomedical and Pharmaceutical Technology and College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350001 (China); Zhang Qiqing, E-mail: zhangqiq@126.com [Institute of Biomedical and Pharmaceutical Technology and College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350001 (China) and Institute of Biomedical Engineering, Chinese Academy of Medical Science, Peking Union Medical College, Tianjin 300192 (China)

2012-06-15

An attempt to improve hydrophilicity and anti-fouling properties of PVDF membranes, a novel amphiphilic zwitterionic copolymer poly(vinylidene fluoride)-graft-poly(sulfobetaine methacrylate) (PVDF-g-PSBMA) was firstly synthesized by atom transfer radical polymerization (ATRP) and used as amphiphilic copolymer additive in the preparation of PVDF membranes. The PVDF-g-PSBMA/PVDF blend membranes were prepared by immersion precipitation process. Fourier transform infrared attenuated reflection spectroscopy (FTIR-ATR) and X-ray photoelectronic spectroscopy (XPS) measurements confirmed that PSBMA brushes from amphiphilic additives were preferentially segregated to membrane-coagulant interface during membrane formation. The morphology of membranes was characterized by scanning electron microscopy (SEM). Water contact angle measurements showed that the surface hydrophilicity of PVDF membranes was improved significantly with the increasing of amphiphilic copolymer PVDF-g-PSBMA in cast solution. Protein static adsorption experiment and dynamic fouling resistance experiment revealed that the surface enrichment of PSBMA brush endowed PVDF blend membrane great improvement of surface anti-fouling ability.

Improved surface property of PVDF membrane with amphiphilic zwitterionic copolymer as membrane additive

International Nuclear Information System (INIS)

Li Jianhua; Li Mizi; Miao Jing; Wang Jiabin; Shao Xisheng; Zhang Qiqing

2012-01-01

An attempt to improve hydrophilicity and anti-fouling properties of PVDF membranes, a novel amphiphilic zwitterionic copolymer poly(vinylidene fluoride)-graft-poly(sulfobetaine methacrylate) (PVDF-g-PSBMA) was firstly synthesized by atom transfer radical polymerization (ATRP) and used as amphiphilic copolymer additive in the preparation of PVDF membranes. The PVDF-g-PSBMA/PVDF blend membranes were prepared by immersion precipitation process. Fourier transform infrared attenuated reflection spectroscopy (FTIR-ATR) and X-ray photoelectronic spectroscopy (XPS) measurements confirmed that PSBMA brushes from amphiphilic additives were preferentially segregated to membrane-coagulant interface during membrane formation. The morphology of membranes was characterized by scanning electron microscopy (SEM). Water contact angle measurements showed that the surface hydrophilicity of PVDF membranes was improved significantly with the increasing of amphiphilic copolymer PVDF-g-PSBMA in cast solution. Protein static adsorption experiment and dynamic fouling resistance experiment revealed that the surface enrichment of PSBMA brush endowed PVDF blend membrane great improvement of surface anti-fouling ability.

Attainability and minimum energy of single-stage membrane and membrane/distillation hybrid processes

KAUST Repository

Alshehri, Ali

2014-12-01

As an energy-efficient separation method, membrane technology has attracted more and more attentions in many challenging separation processes. The attainability and the energy consumption of a membrane process are the two basic fundamental questions that need to be answered. This report aims to use process simulations to find: (1) at what conditions a single-stage membrane process can meet the separation task that is defined by product purity and recovery ratio and (2) what are the most important parameters that determine the energy consumption. To perform a certain separation task, it was found that both membrane selectivity and pressure ratio exhibit a minimum value that is defined only by product purity and recovery ratio. The membrane/distillation hybrid system was used to study the energy consumption. A shortcut method was developed to calculate the minimum practical separation energy (MPSE) of the membrane process and the distillation process. It was found that the MPSE of the hybrid system is only determined by the membrane selectivity and the applied transmembrane pressure ratio in three stages. At the first stage when selectivity is low, the membrane process is not competitive to the distillation process. Adding a membrane unit to a distillation tower will not help in reducing energy. At the second medium selectivity stage, the membrane/distillation hybrid system can help reduce the energy consumption, and the higher the membrane selectivity, the lower is the energy. The energy conservation is further improved as pressure ratio increases. At the third stage when both selectivity and pressure ratio are high, the hybrid system will change to a single-stage membrane unit and this change will cause significant reduction in energy consumption. The energy at this stage keeps decreasing with selectivity at slow rate, but slightly increases with pressure ratio. Overall, the higher the membrane selectivity, the more the energy is saved. Therefore, the two

Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

Science.gov (United States)

Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

2007-08-17

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

Techno-economical evaluation of membrane based biogas upgrading system: A comparison between polymeric membrane and carbon membrane technology

Directory of Open Access Journals (Sweden)

Shamim Haider

2016-10-01

Full Text Available A shift to renewable energy sources will reduce emissions of greenhouse gases and secure future energy supplies. In this context, utilization of biogas will play a prominent role. Focus of this work is upgrading of biogas to fuel quality by membrane separation using a carbon hollow fibre (CHF membrane and compare with a commercially available polymeric membrane (polyimide through economical assessment. CHF membrane modules were prepared for pilot plant testing and performance measured using CO2, O2, N2. The CHF membrane was modified through oxidation, chemical vapour deposition (CVD and reduction process thus tailoring pores for separation and increased performance. The post oxidized and reduced carbon hollow fibres (PORCHFs significantly exceeded CHF performance showing higher CO2 permeance (0.021 m3(STP/m2 h bar and CO2/CH4 selectivity of 246 (5 bar feed vs 50 mbar permeate pressure. The highest performance recorded through experiments (CHF and PORCHF was used as simulation basis. A membrane simulation model was used and interfaced to 8.6 V Aspen HYSYS. A 300 Nm3/h mixture of CO2/CH4 containing 30–50% CO2 at feed pressures 6, 8 and 10 bar, was simulated and process designed to recover 99.5% CH4 with 97.5% purity. Net present value (NPV was calculated for base case and optimal pressure (50 bar for CHF and PORCHF. The results indicated that recycle ratio (recycle/feed ranged from 0.2 to 10, specific energy from 0.15 to 0.8 (kW/Nm3feed and specific membrane area from 45 to 4700 (m2/Nm3feed. The high recycle ratio can create problems during start-up, as it would take long to adjust volumetric flow ratio towards 10. The best membrane separation system employs a three-stage system with polyimide at 10 bar, and a two-stage membrane system with PORCHF membranes at 50 bar with recycle. Considering biomethane price of 0.78 $/Nm3 and a lifetime of 15 years, the techno-economic analysis showed that payback time for

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

International Nuclear Information System (INIS)

Haylett, T.; Thilo, L.

1986-01-01

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

A plant-like proton-pump partnership in the malaria parasite

Energy Technology Data Exchange (ETDEWEB)

Allen, R J.W.; Saliba, K J; Zissis, S; Kirk, K [Australian National University, ACT (Australia)

2001-07-01

Full text: The 'intraerythrocytic' form of the human malaria parasite. Plasmodium falciparum contains an acidic 'digestive vacuole' which is believed to be the main site of haemoglobin degradation, and the major site of action of many antimalarial drugs. The mechanism/s by which this organelle is acidified have not been investigated. In plant cells, the internal acidic vacuole has on its membrane two types of H{sup +}-pumps which contribute to the generation of an acidic pH: a vacuolar-type H{sup +}-ATPase (V-H{sup +}-ATPase) and a vacuolar H{sup +}-pyrophosphatase (V-H{sup +}-PPase). The presence of a V-H{sup +}-ATPase on the digestive vacuole membrane of P. falciparum has been demonstrated by immuno-electron microscopy (J. Biol. Chem. (2000) 275: 34353-34358) but its functional activity on this organelle has not been demonstrated. Two V-H{sup +}-PPase genes have been shown to be expressed in the intraerythrocytic stage of the P. falciparum parasite (Mol. Biochem. Parasitol. (2001) 114: 183-195); however, immunological methods failed to detect either on the parasite digestive vacuole. In this study we use a combination of NMR spectroscopy and fluorescence techniques to show that (i) P. falciparum contains low levels of pyrophosphate, and (ii) that both ATP and pyrophosphate are able to energise the acidification of the parasite's digestive vacuole. We propose that, like many plant cells the digestive vacuole of P. falciparum parasites has, on its membrane, a V-H{sup +}-PPase as well as a V-H{sup +}-ATPaSe, and that both pumps contribute to the pH regulation of this organelle.

Gas separation membranes

Science.gov (United States)

Schell, William J.

1979-01-01

A dry, fabric supported, polymeric gas separation membrane, such as cellulose acetate, is prepared by casting a solution of the polymer onto a shrinkable fabric preferably formed of synthetic polymers such as polyester or polyamide filaments before washing, stretching or calendering (so called griege goods). The supported membrane is then subjected to gelling, annealing, and drying by solvent exchange. During the processing steps, both the fabric support and the membrane shrink a preselected, controlled amount which prevents curling, wrinkling or cracking of the membrane in flat form or when spirally wound into a gas separation element.

Impedance study of membrane dehydration and compression in proton exchange membrane fuel cells

Energy Technology Data Exchange (ETDEWEB)

Le Canut, Jean-Marc; Latham, Ruth; Merida, Walter; Harrington, David A. [Institute for Integrated Energy Systems, University of Victoria, Victoria, British Columbia (Canada)

2009-07-15

Electrochemical impedance spectroscopy (EIS) is used to measure drying and rehydration in proton exchange membrane fuel cells running under load. The hysteresis between forward and backward acquisition of polarization curves is shown to be largely due to changes in the membrane resistance. Drying tests are carried out with hydrogen and simulated reformate (hydrogen and carbon dioxide), and quasi-periodic drying and rehydration conditions are studied. The membrane hydration state is clearly linked to the high-frequency arc in the impedance spectrum, which increases in size for dry conditions indicating an increase in membrane resistance. Changes in impedance spectra as external compression is applied to the cell assembly show that EIS can separate membrane and interfacial effects, and that changes in membrane resistance dominate. Reasons for the presence of a capacitance in parallel with the membrane resistance are discussed. (author)

Cholesteatoma behind an intact tympanic membrane: histopathologic evidence for a tympanic membrane origin.

Science.gov (United States)

Sudhoff, H; Linthicum, F H

2001-07-01

Several theories have been proposed with respect to the origin and pathogenesis of cholesteatoma behind an intact tympanic membrane. The authors describe a case of cholesteatoma behind an intact tympanic membrane in a 71-year-old man with a history of tympanic membrane retraction fixed to the incus without evidence of a perforation. The membrane eventually became detached, and remnants of keratinizing squamous epithelium were found on the incus. Mechanisms such as metaplasia, ectopic epidermis rests, or ingrowth of meatal epidermis have been proposed to explain the pathogenesis of cholesteatoma behind an intact tympanic membrane. These findings, based on temporal bone histopathology, support the role of an acquired epidermal rest. This case report provides evidence that cholesteatoma behind an intact tympanic membrane can be established from a resolved retraction of the pars tensa of the tympanic membrane.

Radiolytic preparation of PFA-g-PVBSA membranes as a polymer electrolyte membrane

Energy Technology Data Exchange (ETDEWEB)

Fei Geng [Department of Chemistry and Materials Engineering, Changshu Institute of Technology, Nansanhuan Road 99, Changshu, Jiangsu 215-500 (China); Hwang, Mi-Lim; Sohn, Joon-Yong; Nho, Young Chang [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong-dong, Jeongeup-si, Jeollabuk-do 580-185 (Korea, Republic of); Shin, Junhwa, E-mail: shinj@kaeri.re.kr [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong-dong, Jeongeup-si, Jeollabuk-do 580-185 (Korea, Republic of)

2012-03-01

In this study, a polymer electrolyte membrane, PFA-g-PVBSA was prepared through the radiation-induced graft copolymerization of vinylbenzyl chloride (VBC) monomer onto a poly(tetrafluoroethylene-co-perfluoropropylvinyl ether) (PFA) film and subsequent sulfonation processes. The IEC values and water uptakes of the prepared membranes increased when increasing the contents of the poly(vinylbenzyl sulfonic acid) (PVBSA) graft polymers in the membranes. Compared with Nafion 212, the degree of grafting (DOG) of membranes of 50% and 70% showed higher proton conductivity with significantly lower methanol permeability. The combination of these properties suggests that the prepared membranes are promising for future application in direct methanol fuel cells.

The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus.

Science.gov (United States)

Lee, Myoung Hui; Yoo, Yun-Joo; Kim, Dae Heon; Hanh, Nguyen Hong; Kwon, Yun; Hwang, Inhwan

2017-07-01

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis ( Arabidopsis thaliana ) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4 , atpra1.f4 , was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na + /K + -ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA : AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus. © 2017 American Society of Plant Biologists. All Rights Reserved.

Specific ion effects on membrane potential and the permselectivity of ion exchange membranes

KAUST Repository

Geise, Geoffrey M.

2014-08-26

© the Partner Organisations 2014. Membrane potential and permselectivity are critical parameters for a variety of electrochemically-driven separation and energy technologies. An electric potential is developed when a membrane separates electrolyte solutions of different concentrations, and a permselective membrane allows specific species to be transported while restricting the passage of other species. Ion exchange membranes are commonly used in applications that require advanced ionic electrolytes and span technologies such as alkaline batteries to ammonium bicarbonate reverse electrodialysis, but membranes are often only characterized in sodium chloride solutions. Our goal in this work was to better understand membrane behaviour in aqueous ammonium bicarbonate, which is of interest for closed-loop energy generation processes. Here we characterized the permselectivity of four commercial ion exchange membranes in aqueous solutions of sodium chloride, ammonium chloride, sodium bicarbonate, and ammonium bicarbonate. This stepwise approach, using four different ions in aqueous solution, was used to better understand how these specific ions affect ion transport in ion exchange membranes. Characterization of cation and anion exchange membrane permselectivity, using these ions, is discussed from the perspective of the difference in the physical chemistry of the hydrated ions, along with an accompanying re-derivation and examination of the basic equations that describe membrane potential. In general, permselectivity was highest in sodium chloride and lowest in ammonium bicarbonate solutions, and the nature of both the counter- and co-ions appeared to influence measured permselectivity. The counter-ion type influences the binding affinity between counter-ions and polymer fixed charge groups, and higher binding affinity between fixed charge sites and counter-ions within the membrane decreases the effective membrane charge density. As a result permselectivity decreases. The

In-situcross-linked PVDF membranes with enhanced mechanical durability for vacuum membrane distillation

KAUST Repository

Zuo, Jian; Chung, Neal Tai-Shung

2016-01-01

A novel and effective one-step method has been demonstrated to fabricate cross-linked polyvinylidene fluoride (PVDF) membranes with better mechanical properties and flux for seawater desalination via vacuum membrane distillation (VMD). This method involves the addition of two functional nonsolvent additives; namely, water and ethylenediamine (EDA), into the polymer casting solution. The former acts as a pore forming agent, while the latter performs as a cross-linking inducer. The incorporation of water tends to increase membrane flux via increasing porosity and pore size but sacrifices membrane mechanical properties. Conversely, the presence of EDA enhances membrane mechanical properties through in-situ cross-linking reaction. Therefore, by synergistically combining the effects of both functional additives, the resultant PVDF membranes have shown good MD performance and mechanical properties simultaneously. The parameters that affect the cross-link reaction and membrane mechanical properties such as reaction duration and EDA concentration have been systematically studied. The membranes cast from an optimal reaction condition comprising 0.8 wt % EDA and 3-hour reaction not only shows a 40% enhancement in membrane Young's Modulus compared to the one without EDA but also achieves a good VMD flux of 43.6 L/m2-h at 60°C. This study may open up a totally new approach to design next-generation high performance MD membranes. © 2016 American Institute of Chemical Engineers AIChE J, 62: 4013–4022, 2016

In-situcross-linked PVDF membranes with enhanced mechanical durability for vacuum membrane distillation

KAUST Repository

Zuo, Jian

2016-05-12

A novel and effective one-step method has been demonstrated to fabricate cross-linked polyvinylidene fluoride (PVDF) membranes with better mechanical properties and flux for seawater desalination via vacuum membrane distillation (VMD). This method involves the addition of two functional nonsolvent additives; namely, water and ethylenediamine (EDA), into the polymer casting solution. The former acts as a pore forming agent, while the latter performs as a cross-linking inducer. The incorporation of water tends to increase membrane flux via increasing porosity and pore size but sacrifices membrane mechanical properties. Conversely, the presence of EDA enhances membrane mechanical properties through in-situ cross-linking reaction. Therefore, by synergistically combining the effects of both functional additives, the resultant PVDF membranes have shown good MD performance and mechanical properties simultaneously. The parameters that affect the cross-link reaction and membrane mechanical properties such as reaction duration and EDA concentration have been systematically studied. The membranes cast from an optimal reaction condition comprising 0.8 wt % EDA and 3-hour reaction not only shows a 40% enhancement in membrane Young\\'s Modulus compared to the one without EDA but also achieves a good VMD flux of 43.6 L/m2-h at 60°C. This study may open up a totally new approach to design next-generation high performance MD membranes. © 2016 American Institute of Chemical Engineers AIChE J, 62: 4013–4022, 2016

Fabrication of bioinspired composite nanofiber membranes with robust superhydrophobicity for direct contact membrane distillation.

Science.gov (United States)

Liao, Yuan; Wang, Rong; Fane, Anthony G

2014-06-03

The practical application of membrane distillation (MD) for water purification is hindered by the absence of desirable membranes that can fulfill the special requirements of the MD process. Compared to the membranes fabricated by other methods, nanofiber membranes produced by electrospinning are of great interest due to their high porosity, low tortuosity, large surface pore size, and high surface hydrophobicity. However, the stable performance of the nanofiber membranes in the MD process is still unsatisfactory. Inspired by the unique structure of the lotus leaf, this study aimed to develop a strategy to construct superhydrophobic composite nanofiber membranes with robust superhydrophobicity and high porosity suitable for use in MD. The newly developed membrane consists of a superhydrophobic silica-PVDF composite selective skin formed on a polyvinylidene fluoride (PVDF) porous nanofiber scaffold via electrospinning. This fabrication method could be easily scaled up due to its simple preparation procedures. The effects of silica diameter and concentration on membrane contact angle, sliding angle, and MD performance were investigated thoroughly. For the first time, the direct contact membrane distillation (DCMD) tests demonstrate that the newly developed membranes are able to present stable high performance over 50 h of testing time, and the superhydrophobic selective layer exhibits excellent durability in ultrasonic treatment and a continuous DCMD test. It is believed that this novel design strategy has great potential for MD membrane fabrication.

Effect of Plasma Membrane Semipermeability in Making the Membrane Electric Double Layer Capacitances Significant.

Science.gov (United States)

Sinha, Shayandev; Sachar, Harnoor Singh; Das, Siddhartha

2018-01-30

Electric double layers (or EDLs) formed at the membrane-electrolyte interface (MEI) and membrane-cytosol interface (MCI) of a charged lipid bilayer plasma membrane develop finitely large capacitances. However, these EDL capacitances are often much larger than the intrinsic capacitance of the membrane, and all of these capacitances are in series. Consequently, the effect of these EDL capacitances in dictating the overall membrane-EDL effective capacitance C eff becomes negligible. In this paper, we challenge this conventional notion pertaining to the membrane-EDL capacitances. We demonstrate that, on the basis of the system parameters, the EDL capacitance for both the permeable and semipermeable membranes can be small enough to influence C eff . For the semipermeable membranes, however, this lowering of the EDL capacitance can be much larger, ensuring a reduction of C eff by more than 20-25%. Furthermore, for the semipermeable membranes, the reduction in C eff is witnessed over a much larger range of system parameters. We attribute such an occurrence to the highly nonintuitive electrostatic potential distribution associated with the recently discovered phenomena of charge-inversion-like electrostatics and the attainment of a positive zeta potential at the MCI for charged semipermeable membranes. We anticipate that our findings will impact the quantification and the identification of a large number of biophysical phenomena that are probed by measuring the plasma membrane capacitance.

Isolation of plasma membrane-associated membranes from rat liver.

Science.gov (United States)

Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R

2014-02-01

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

DEFF Research Database (Denmark)

Javanainen, Matti; Martinez-Seara, Hector; Metzler, Ralf

2017-01-01

of being protein-poor, native cell membranes are extremely crowded with proteins. On the basis of extensive molecular simulations, we here demonstrate that protein crowding of the membrane at physiological levels leads to deviations from the SD relation and to the emergence of a stronger Stokes......-like dependence D ∝ 1/R. We propose that this 1/R law mainly arises due to geometrical factors: smaller proteins are able to avoid confinement effects much better than their larger counterparts. The results highlight that the lateral dynamics in the crowded setting found in native membranes is radically different......The lateral diffusion of embedded proteins along lipid membranes in protein-poor conditions has been successfully described in terms of the Saffman-Delbrück (SD) model, which predicts that the protein diffusion coefficient D is weakly dependent on its radius R as D ∝ ln(1/R). However, instead...

Membrane Bioreactor (MBR) Technology for Wastewater Treatment and Reclamation: Membrane Fouling.

Science.gov (United States)

Iorhemen, Oliver Terna; Hamza, Rania Ahmed; Tay, Joo Hwa

2016-06-15

The membrane bioreactor (MBR) has emerged as an efficient compact technology for municipal and industrial wastewater treatment. The major drawback impeding wider application of MBRs is membrane fouling, which significantly reduces membrane performance and lifespan, resulting in a significant increase in maintenance and operating costs. Finding sustainable membrane fouling mitigation strategies in MBRs has been one of the main concerns over the last two decades. This paper provides an overview of membrane fouling and studies conducted to identify mitigating strategies for fouling in MBRs. Classes of foulants, including biofoulants, organic foulants and inorganic foulants, as well as factors influencing membrane fouling are outlined. Recent research attempts on fouling control, including addition of coagulants and adsorbents, combination of aerobic granulation with MBRs, introduction of granular materials with air scouring in the MBR tank, and quorum quenching are presented. The addition of coagulants and adsorbents shows a significant membrane fouling reduction, but further research is needed to establish optimum dosages of the various coagulants/adsorbents. Similarly, the integration of aerobic granulation with MBRs, which targets biofoulants and organic foulants, shows outstanding filtration performance and a significant reduction in fouling rate, as well as excellent nutrients removal. However, further research is needed on the enhancement of long-term granule integrity. Quorum quenching also offers a strong potential for fouling control, but pilot-scale testing is required to explore the feasibility of full-scale application.

A Quaternary Polybenzimidazole Membrane for Intermediate Temperature Polymer Electrolyte Membrane Fuel Cells

DEFF Research Database (Denmark)

Xu, C.; Scott, K.; Li, Qingfeng

2013-01-01

at 150 °C with the PA acid loading level of 3.5 PRU (amount of H3PO4 per repeat unit of polymer QPBI). The QPBI membrane was characterized in terms of composition, structure and morphology by NMR, FTIR, SEM, and EDX. The fuel cell performance with the membrane gave peak power densities of 440 and 240 m......A quaternary ammonium polybenzimidazole (QPBI) membrane was synthesized for applications in intermediate temperature (100–200 °C) hydrogen fuel cells. The QPBI membrane was imbibed with phosphoric acid to provide suitable proton conductivity. The proton conductivity of the membrane was 0.051 S cm–1......W cm–2 using oxygen and air, respectively, at 175 °C....

Membrane fusion and exocytosis.

Science.gov (United States)

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

Science.gov (United States)

Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

2016-09-01

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair

DEFF Research Database (Denmark)

Boye, Theresa Louise; Maeda, Kenji; Pezeshkian, Weria

2017-01-01

Efficient cell membrane repair mechanisms are essential for maintaining membrane integrity and thus for cell life. Here we show that the Ca2+- and phospholipid-binding proteins annexin A4 and A6 are involved in plasma membrane repair and needed for rapid closure of micron-size holes. We demonstrate...... that annexin A4 binds to artificial membranes and generates curvature force initiated from free edges, whereas annexin A6 induces constriction force. In cells, plasma membrane injury and Ca2+ influx recruit annexin A4 to the vicinity of membrane wound edges where its homo-trimerization leads to membrane...... that induction of curvature force around wound edges is an early key event in cell membrane repair....

PVDF hollow fiber and nanofiber membranes for fresh water reclamation using membrane distillation

KAUST Repository

Francis, Lijo; Ghaffour, NorEddine; Alsaadi, Ahmad Salem; Nunes, Suzana Pereira; Amy, Gary L.

2013-01-01

Polyvinylidene fluoride hollow fiber and nanofibrous membranes are engineered and successfully fabricated using dry-jet wet spinning and electrospinning techniques, respectively. Fabricated membranes are characterized for their morphology, average pore size, pore size distribution, nanofiber diameter distribution, thickness, and water contact angle. Direct contact membrane distillation (DCMD) performances of the fabricated membranes have been investigated using a locally designed and fabricated, fully automated MD bench scale unit and DCMD module. Electrospun nanofibrous membranes showed a water flux as high as 36 L m-2 h-1 whereas hollow fiber membranes showed a water flux of 31.6 L m-2 h-1, at a feed inlet temperature of 80 °C and at a permeate inlet temperature of 20 °C.

PVDF hollow fiber and nanofiber membranes for fresh water reclamation using membrane distillation

KAUST Repository

Francis, Lijo

2013-11-26

Polyvinylidene fluoride hollow fiber and nanofibrous membranes are engineered and successfully fabricated using dry-jet wet spinning and electrospinning techniques, respectively. Fabricated membranes are characterized for their morphology, average pore size, pore size distribution, nanofiber diameter distribution, thickness, and water contact angle. Direct contact membrane distillation (DCMD) performances of the fabricated membranes have been investigated using a locally designed and fabricated, fully automated MD bench scale unit and DCMD module. Electrospun nanofibrous membranes showed a water flux as high as 36 L m-2 h-1 whereas hollow fiber membranes showed a water flux of 31.6 L m-2 h-1, at a feed inlet temperature of 80 °C and at a permeate inlet temperature of 20 °C.

Chelating polymeric membranes

KAUST Repository

Peinemann, Klaus-Viktor

2015-01-22

The present application offers a solution to the current problems associated with recovery and recycling of precious metals from scrap material, discard articles, and other items comprising one or more precious metals. The solution is premised on a microporous chelating polymeric membrane. Embodiments include, but are not limited to, microporous chelating polymeric membranes, device comprising the membranes, and methods of using and making the same.

Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species.

Science.gov (United States)

Carmona-Salazar, Laura; El Hafidi, Mohammed; Gutiérrez-Nájera, Nora; Noyola-Martínez, Liliana; González-Solís, Ariadna; Gavilanes-Ruíz, Marina

2015-01-01

It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical-chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

Science.gov (United States)

Cheng, Chi-Yuan; Han, Songi

2013-01-01

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

DEFF Research Database (Denmark)

Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

2006-01-01

A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials...

Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

Science.gov (United States)

Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

2018-02-20

Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

Sweep gas membrane distillation in a membrane contactor with metallic hollow fibers

NARCIS (Netherlands)

Shukla, Sushumna; Benes, Nieck Edwin; Vankelecom, I.F.J.; Mericq, J.P.; Belleville, M.P.; Hengl, N.; Sanchez Marcano, Jose

2015-01-01

This work revolves around the use of porous metal hollow fibers in membrane distillation. Various stages are covered, starting from membrane synthesis up to the testing of a pilot scale membrane module. Mechanically stable metal hollow fibers have been synthesized by phase inversion of a stainless

Flavonoid-membrane Interactions: A Protective Role of Flavonoids at the Membrane Surface?

Directory of Open Access Journals (Sweden)

Patricia I. Oteiza

2005-01-01

Full Text Available Flavonoids can exert beneficial health effects through multiple mechanisms. In this paper, we address the important, although not fully understood, capacity of flavonoids to interact with cell membranes. The interactions of polyphenols with bilayers include: (a the partition of the more non-polar compounds in the hydrophobic interior of the membrane, and (b the formation of hydrogen bonds between the polar head groups of lipids and the more hydrophilic flavonoids at the membrane interface. The consequences of these interactions are discussed. The induction of changes in membrane physical properties can affect the rates of membrane lipid and protein oxidation. The partition of certain flavonoids in the hydrophobic core can result in a chain breaking antioxidant activity. We suggest that interactions of polyphenols at the surface of bilayers through hydrogen bonding, can act to reduce the access of deleterious molecules (i.e. oxidants, thus protecting the structure and function of membranes.

Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

Science.gov (United States)

Zhu, Xiaobiao; Gong, Huiling; He, Qunyan; Zeng, Zixian; Busse, James S; Jin, Weiwei; Bethke, Paul C; Jiang, Jiming

2016-02-01

Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Clinical features of progressive vacuolar hepatopathy in Scottish Terriers with and without hepatocellular carcinoma: 114 cases (1980-2013).

Science.gov (United States)

Cortright, Catherine C; Center, Sharon A; Randolph, John F; McDonough, Sean P; Fecteau, Kellie A; Warner, Karen L; Chiapella, Ann M; Pierce, Rhonda L; Graham, A Heather; Wall, Linda J; Heidgerd, John H; Degen, Melisa A; Lucia, Patricia A; Erb, Hollis N

2014-10-01

To characterize signalment, clinical features, clinicopathologic variables, hepatic ultrasonographic characteristics, endocrinologic profiles, treatment response, and age at death of Scottish Terriers with progressive vacuolar hepatopathy (VH) with or without hepatocellular carcinoma (HCC). Retrospective case series. 114 Scottish Terriers with progressive VH. Electronic databases from 1980 to 2013 were searched for adult (age > 1 year) Scottish Terriers with histopathologic diagnoses of diffuse glycogen-like VH. Available sections of liver specimens were histologically reevaluated to confirm diffuse VH with or without HCC; 8 dogs with HCC only had neoplastic tissue available. Physical examination, clinicopathologic, treatment, and survival data were obtained. 39 of 114 (34%) dogs with VH had HCC detected at surgery or necropsy or by abdominal ultrasonography. Histologic findings indicated that HCC was seemingly preceded by dysplastic hepatocellular foci. No significant differences were found in clinicopathologic variables or age at death between VH-affected dogs with or without HCC. Fifteen of 26 (58%) dogs with high hepatic copper concentrations had histologic features consistent with copper-associated hepatopathy. Although signs consistent with hyperadrenocorticism were observed in 40% (46/114) of dogs, definitive diagnosis was inconsistently confirmed. Assessment of adrenal sex hormone concentrations before and after ACTH administration identified high progesterone and androstenedione concentrations in 88% (22/25) and 80% (20/25) of tested dogs, respectively. Results suggested that VH in Scottish Terriers may be linked to adrenal steroidogenesis and a predisposition to HCC. In dogs with VH, frequent serum biochemical analysis and ultrasonographic surveillance for early tumor detection are recommended.

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane

OpenAIRE

Doudová, Lenka

2017-01-01

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an importa...

Radio-chemical applications of functionalized membranes

International Nuclear Information System (INIS)

Pandey, Ashok K.

2011-01-01

Functionalized polymer membranes have many potential applications as they are task specific. We have developed many functionalized membranes like polymer inclusion membranes, pore-filled membranes and nano-membranes. Radiotracers and other methods have been used to understand the diffusional-transport properties of the Nafion-117 membrane as well as home-made membranes. These membranes have been used to develop novel analytical and separation methods for toxic metal ions and radionuclides. In this talk, an overview of our work on functionalized membrane is presented. (author)

A study for the research trends of membranes for proton exchange membrane fuel cells

International Nuclear Information System (INIS)

Sener, T.

2004-01-01

'Full text:' A single PEM fuel cell is comprised of a membrane electrode assembly, two bipolar plates and two fields. Membrane electrode assembly is the basic component of PEM fuel cell due to its cost and function, and it consists a membrane sandwiched between two electrocatalyst layers/electrodes and two gas diffusion layers. Increasing the PEM fuel cell operation temperature from 80 o C to 150-200 o C will prevent electrocatalysts CO poisoning and increase the fuel cell performance. Therefore, membranes must have chemical and mechanical resistance and must keep enough water at high temperatures. The aim of membrane studies through fuel cell commercialization is to produce a less expensive thin membrane with high operation temperature, chemical and mechanical resistance and water adsorption capacity. Within this frame, alternative membrane materials, membrane electrode assembly manufacture and evaluation methods are being studied. In this paper, recent studies are reviewed to give a conclusion for research trends. (author)

Analysis of Protein-Membrane Interactions

DEFF Research Database (Denmark)

Kemmer, Gerdi Christine

Cellular membranes are complex structures, consisting of hundreds of different lipids and proteins. These membranes act as barriers between distinct environments, constituting hot spots for many essential functions of the cell, including signaling, energy conversion, and transport. These functions....... Discovered interactions were then probed on the level of the membrane using liposome-based assays. In the second part, a transmembrane protein was investigated. Assays to probe activity of the plasma membrane ATPase (Arabidopsis thaliana H+ -ATPase isoform 2 (AHA2)) in single liposomes using both giant...... are implemented by soluble proteins reversibly binding to, as well as by integral membrane proteins embedded in, cellular membranes. The activity and interaction of these proteins is furthermore modulated by the lipids of the membrane. Here, liposomes were used as model membrane systems to investigate...

Improved antifouling potential of polyether sulfone polymeric membrane containing silver nanoparticles: self-cleaning membranes.

Science.gov (United States)

Rana, Sidra; Nazar, Umair; Ali, Jafar; Ali, Qurat Ul Ain; Ahmad, Nasir M; Sarwar, Fiza; Waseem, Hassan; Jamil, Syed Umair Ullah

2018-06-01

A new strategy to enhance the antifouling potential of polyether sulfone (PES) membrane is presented. Chemically synthesized silver nanoparticles (AgNPs) were used to prepare a mixed-matrix PES membrane by the phase inversion technique. Primarily, AgNPs synthesis was confirmed by surface plasmon resonance at 410-430 nm using UV-Visible spectroscopy. X-ray diffraction analysis revealed that AgNPs were crystalline with a diameter of 21 ± 2 nm. Furthermore, PES membranes were characterized by energy dispersive X-ray spectroscopy to confirm the incorporation of AgNPs in membranes. Hydrophilicity of the membranes was enhanced, whereas roughness, mechanical strength and biofouling were relatively reduced after embedding the AgNPs. Antibacterial potential of AgNPs was evaluated for E. coli in the disc diffusion and colony-forming unit (CFU) count method. All of the membranes were assessed for antifouling activity by filtering a control dilution (10 6  CFU/ml) of E. coli and by counting CFU. Anti-biofouling activity of the membrane was observed with different concentrations of AgNPs. Maximum reduction (66%) was observed in membrane containing 1.5% of AgNPs. The addition of antibiotic ceftriaxone enhanced the antibacterial effect of AgNPs in PES membranes. Our practicable antifouling strategy may be applied to other polymeric membranes which may pave the new way to achieve sustainable and self-cleaning membrane reactors on large scale.

Membrane adsorber for endotoxin removal

Directory of Open Access Journals (Sweden)

Karina Moita de Almeida

Full Text Available ABSTRACT The surface of flat-sheet nylon membranes was modified using bisoxirane as the spacer and polyvinyl alcohol as the coating polymer. The amino acid histidine was explored as a ligand for endotoxins, aiming at its application for endotoxin removal from aqueous solutions. Characterization of the membrane adsorber, analysis of the depyrogenation procedures and the evaluation of endotoxin removal efficiency in static mode are discussed. Ligand density of the membranes was around 7 mg/g dry membrane, allowing removal of up to 65% of the endotoxins. The performance of the membrane adsorber prepared using nylon coated with polyvinyl alcohol and containing histidine as the ligand proved superior to other membrane adsorbers reported in the literature. The lack of endotoxin adsorption on nylon membranes without histidine confirmed that endotoxin removal was due to the presence of the ligand at the membrane surface. Modified membranes were highly stable, exhibiting a lifespan of approximately thirty months.

The ESCRT regulator Did2 maintains the balance between long-distance endosomal transport and endocytic trafficking.

Directory of Open Access Journals (Sweden)

Carl Haag

2017-04-01

Full Text Available In highly polarised cells, like fungal hyphae, early endosomes function in both endocytosis as well as long-distance transport of various cargo including mRNA and protein complexes. However, knowledge on the crosstalk between these seemingly different trafficking processes is scarce. Here, we demonstrate that the ESCRT regulator Did2 coordinates endosomal transport in fungal hyphae of Ustilago maydis. Loss of Did2 results in defective vacuolar targeting, less processive long-distance transport and abnormal shuttling of early endosomes. Importantly, the late endosomal protein Rab7 and vacuolar protease Prc1 exhibit increased shuttling on these aberrant endosomes suggesting defects in endosomal maturation and identity. Consistently, molecular motors fail to attach efficiently explaining the disturbed processive movement. Furthermore, the endosomal mRNP linker protein Upa1 is hardly present on endosomes resulting in defects in long-distance mRNA transport. In conclusion, the ESCRT regulator Did2 coordinates precise maturation of endosomes and thus provides the correct membrane identity for efficient endosomal long-distance transport.

Drug-model membrane interactions

International Nuclear Information System (INIS)

Deniz, Usha K.

1994-01-01

In the present day world, drugs play a very important role in medicine and it is necessary to understand their mode of action at the molecular level, in order to optimise their use. Studies of drug-biomembrane interactions are essential for gaining such as understanding. However, it would be prohibitively difficult to carry out such studies, since biomembranes are highly complex systems. Hence, model membranes (made up of these lipids which are important components of biomembranes) of varying degrees of complexity are used to investigate drug-membrane interactions. Bio- as well as model-membranes undergo a chain melting transition when heated, the chains being in a disordered state above the transition point, T CM . This transition is of physiological importance since biomembranes select their components such that T CM is less than the ambient temperature but not very much so, so that membrane flexibility is ensured and porosity, avoided. The influence of drugs on the transition gives valuable clues about various parameters such as the location of the drug in the membrane. Deep insights into drug-membrane interactions are obtained by observing the effect of drugs on membrane structure and the mobilities of the various groups in lipids, near T CM . Investigation of such changes have been carried out with several drugs, using techniques such as DSC, XRD and NMR. The results indicate that the drug-membrane interaction not only depends on the nature of drug and lipids but also on the form of the model membrane - stacked bilayer or vesicles. The light that these results shed on the nature of drug-membrane interactions is discussed. (author). 13 refs., 13 figs., 1 tab

Counter-current membrane reactor for WGS process: Membrane design

Energy Technology Data Exchange (ETDEWEB)

Piemonte, Vincenzo; Favetta, Barbara [Department of Chemical Engineering Materials and Environment, University of Rome ' ' La Sapienza' ' , via Eudossiana 18, 00184 Rome (Italy); De Falco, Marcello [Faculty of Engineering, University Campus Bio-Medico of Rome, via Alvaro del Portillo 21, 00128 Rome (Italy); Basile, Angelo [CNR-ITM, c/o University of Calabria, Via Pietro Bucci, Cubo 17/C, 87030 Rende (CS) (Italy)

2010-11-15

Water gas shift (WGS) is a thermodynamically limited reaction which has to operate at low temperatures, reducing kinetics rate and increasing the amount of catalyst required to reach valuable CO conversions. It has been widely demonstrated that the integration of hydrogen selective membranes is a promising way to enhance WGS reactors performance: a Pd-based MR operated successfully overcoming the thermodynamic constraints of a traditional reactor thanks to the removal of hydrogen from reaction environment. In the first part of a MR, the H{sub 2} partial pressure starts from a minimum value since the reaction has not started. As a consequence, if the carrier gas in the permeation zone is sent in counter-current, which is the most efficient configuration, in the first reactor section the H{sub 2} partial pressure in reaction zone is low while in the permeation zone is high, potentially implying back permeation. This means a bad utilization of the first part of the membrane area and thus, a worsening of the MR performance with lower H{sub 2} recovery and lower CO conversion with respect to the case in which the whole selective surface is properly used. To avoid this problem different MR configurations were evaluated by a 1-D pseudo-homogeneous model, validated with WGS industrial data reported in scientific literature. It was demonstrated that the permeated H{sub 2} flow rate per membrane surface, i.e. the membrane flux, strongly improves if selective membrane is placed only in the second part of the reactor: in fact, if the membrane is placed at L{sub m}/L{sub tot} = 0.5, the membrane flux is 0.2 kmol/(m{sup 2}h) about, if it is placed along all reactor tube (L{sub m}/L{sub tot} = 1), flux is 0.05 kmol/(m{sup 2}h). The effect of the L/D reactor ratio and of the reactor wall temperature on the CO conversion were also assessed. (author)

Gas separation with membranes

International Nuclear Information System (INIS)

Schulz, G.; Michele, H.; Werner, U.

1982-01-01

Gas separation with membranes has already been tested in numerous fields of application, e.g. uranium enrichment of H 2 separation. In many of these processes the mass transfer units, so-called permeators, have to be connected in tandem in order to achieve high concentrations. A most economical operating method provides for each case an optimization of the cascades with regard to the membrane materials, construction and design of module. By utilization of the concentration gradient along the membrane a new process development has been accomplished - the continuously operating membrane rectification unit. Investment and operating costs can be reduced considerably for a number of separating processes by combining a membrane rectification unit with a conventional recycling cascade. However, the new procedure requires that the specifications for the module construction, flow design, and membrane properties be reconsidered. (orig.) [de

Fetal membrane healing after spontaneous and iatrogenic membrane rupture: A review of current evidence

OpenAIRE

Devlieger, R.; Millar, L. K.; Bryant-Greenwood, G.; Lewi, L.; Deprest, J. A.

2006-01-01

In view of the important protective role of the fetal membranes, wound sealing, tissue regeneration, or wound healing could be life saving in cases of preterm premature rupture of the membranes. Although many investigators are studying the causes of preterm premature rupture of membranes, the emphasis has not been on the wound healing capacity of the fetal membranes. In this review, the relevant literature on the pathophysiologic condition that leads to preterm premature rupture of membranes ...

Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

Energy Technology Data Exchange (ETDEWEB)

Straatsma, TP

2006-12-01

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

In-situ membrane hydration measurement of proton exchange membrane fuel cells

Science.gov (United States)

Lai, Yeh-Hung; Fly, Gerald W.; Clapham, Shawn

2015-01-01

Achieving proper membrane hydration control is one of the most critical aspects of PEM fuel cell development. This article describes the development and application of a novel 50 cm2 fuel cell device to study the in-situ membrane hydration by measuring the through-thickness membrane swelling via an array of linear variable differential transducers. Using this setup either as an air/air (dummy) cell or as a hydrogen/air (operating) cell, we performed a series of hydration and dehydration experiments by cycling the RH of the inlet gas streams at 80 °C. From the linear relationship between the under-the-land swelling and the over-the-channel water content, the mechanical constraint within the fuel cell assembly can suppress the membrane water uptake by 11%-18%. The results from the air/air humidity cycling test show that the membrane can equilibrate within 120 s for all RH conditions and that membrane can reach full hydration at a RH higher than 140% in spite of the use of a liquid water impermeable Carbel MP30Z microporous layer. This result confirms that the U.S. DOE's humidity cycling mechanical durability protocol induces sufficient humidity swings to maximize hygrothermal mechanical stresses. This study shows that the novel experimental technique can provide a robust and accurate means to study the in-situ hydration of thin membranes subject to a wide range of fuel cell conditions.

Membrane Bioreactor (MBR Technology for Wastewater Treatment and Reclamation: Membrane Fouling

Directory of Open Access Journals (Sweden)

Oliver Terna Iorhemen

2016-06-01

Full Text Available The membrane bioreactor (MBR has emerged as an efficient compact technology for municipal and industrial wastewater treatment. The major drawback impeding wider application of MBRs is membrane fouling, which significantly reduces membrane performance and lifespan, resulting in a significant increase in maintenance and operating costs. Finding sustainable membrane fouling mitigation strategies in MBRs has been one of the main concerns over the last two decades. This paper provides an overview of membrane fouling and studies conducted to identify mitigating strategies for fouling in MBRs. Classes of foulants, including biofoulants, organic foulants and inorganic foulants, as well as factors influencing membrane fouling are outlined. Recent research attempts on fouling control, including addition of coagulants and adsorbents, combination of aerobic granulation with MBRs, introduction of granular materials with air scouring in the MBR tank, and quorum quenching are presented. The addition of coagulants and adsorbents shows a significant membrane fouling reduction, but further research is needed to establish optimum dosages of the various coagulants/adsorbents. Similarly, the integration of aerobic granulation with MBRs, which targets biofoulants and organic foulants, shows outstanding filtration performance and a significant reduction in fouling rate, as well as excellent nutrients removal. However, further research is needed on the enhancement of long-term granule integrity. Quorum quenching also offers a strong potential for fouling control, but pilot-scale testing is required to explore the feasibility of full-scale application.

Parallel artificial liquid membrane extraction

DEFF Research Database (Denmark)

Gjelstad, Astrid; Rasmussen, Knut Einar; Parmer, Marthe Petrine

2013-01-01

This paper reports development of a new approach towards analytical liquid-liquid-liquid membrane extraction termed parallel artificial liquid membrane extraction. A donor plate and acceptor plate create a sandwich, in which each sample (human plasma) and acceptor solution is separated by an arti......This paper reports development of a new approach towards analytical liquid-liquid-liquid membrane extraction termed parallel artificial liquid membrane extraction. A donor plate and acceptor plate create a sandwich, in which each sample (human plasma) and acceptor solution is separated...... by an artificial liquid membrane. Parallel artificial liquid membrane extraction is a modification of hollow-fiber liquid-phase microextraction, where the hollow fibers are replaced by flat membranes in a 96-well plate format....

Epoxides cross-linked hexafluoropropylidene polybenzimidazole membranes for application as high temperature proton exchange membranes

International Nuclear Information System (INIS)

Yang, Jingshuai; Xu, Yixin; Liu, Peipei; Gao, Liping; Che, Quantong; He, Ronghuan

2015-01-01

Covalently cross-linked hexafluoropropylidene polybenzimidazole (F 6 PBI) was prepared and used to fabricate high temperature proton exchange membranes with enhanced mechanical strength against thermoplastic distortion. Three different epoxides, i.e. bisphenol A diglycidyl ether (R 1 ), bisphenol A propoxylate diglycidyl ether (R 2 ) and poly(ethylene glycol) diglycidyl ether (R 3 ), were chosen as the cross-linkers to investigate the influence of their structures on the properties of the cross-linked F 6 PBI membranes. All the cross-linked F 6 PBI membranes displayed excellent stability towards the radical oxidation. Comparing with the pure F 6 PBI membrane, the cross-linked F 6 PBI membranes showed high acid doping level but less swelling after doping phosphoric acid at elevated temperatures. The mechanical strength at 130 °C was improved from 0.4 MPa for F 6 PBI membrane to a range of 0.8–2.0 MPa for the cross-linked F 6 PBI membranes with an acid doping level as high as around 14, especially for that crosslinking with the epoxide (R 3 ), which has a long linear structure of alkyl ether. The proton conductivity of the cross-linked membranes was increased accordingly due to the high acid doping levels. Fuel cell tests demonstrated the technical feasibility of the acid doped cross-linked F 6 PBI membranes for high temperature proton exchange membrane fuel cells

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity.

Science.gov (United States)

Morigaki, Kenichi; Tanimoto, Yasushi

2018-03-14

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates. Copyright © 2018 Elsevier B.V. All rights reserved.

Membrane transport of anandamide through resealed human red blood cell membranes

DEFF Research Database (Denmark)

Bojesen, I.N.; Hansen, Harald S.

2005-01-01

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments...... at 0°C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 ± 0.023, and the rate constant...... of unidirectional flux from inside to outside is 0.361 ± 0.023 s. The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]) increases with the square root of [BSA] in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane...

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

Science.gov (United States)

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

2017-01-01

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

Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

Directory of Open Access Journals (Sweden)

Paola Fabrizio

2010-07-01

Full Text Available The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Facile preparation of salt-tolerant anion-exchange membrane adsorber using hydrophobic membrane as substrate.

Science.gov (United States)

Fan, Jinxin; Luo, Jianquan; Chen, Xiangrong; Wan, Yinhua

2017-03-24

In this study, a polyvinylidene fluoride (PVDF) hydrophobic membrane with high mechanical property was used as substrate to prepare salt-tolerant anion-exchange (STAE) membrane adsorber. Effective hydrophilization and functionalization of PVDF membrane was realized via polydopamine (PDA) deposition, thus overcoming the drawbacks of hydrophobic substrates including poor water permeability, inert property as well as severe non-specific adsorption. The following polyallylamine (PAH) coupling was carried out at pH 10.0, where unprotonated amine groups on PAH chains were more prone to couple with PDA. This membrane adsorber could remain 75% of protein binding capacity when NaCl concentration increased from 0 to 150mM, while its protein binding capacity was independent of flow rate from 10 to 100 membrane volume (MV)/min due to its high mechanical strength (tensile strength: 43.58±2.30MPa). With 200mM NaCl addition at pH 7.5, high purity (above 99%) and high recovery (almost 100%) of Immunoglobulin G (IgG) were obtained when using the STAE membrane adsorber to separate IgG/human serum albumin (HSA) mixture, being similar to that without NaCl at pH 6.0 (both under the flow rate of 10-100MV/min). Finally, the reliable reusability was confirmed by five reuse cycles of protein binding and elution operations. In comparison with commercial membrane adsorber, the new membrane adsorber exhibited a better mechanical property, higher IgG polishing efficiency and reusability, while the protein binding capacity was lower due to less NH 2 density on the membrane. The outcome of this work not only offers a facile and effective approach to prepare membrane adsorbers based on hydrophobic membranes, but also demonstrates great potential of this new designed STAE membrane adsorbers for efficient monoclonal antibody (mAb) polishing. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanics of Lipid Bilayer Membranes

Science.gov (United States)

Powers, Thomas R.

All cells have membranes. The plasma membrane encapsulates the cell's interior, acting as a barrier against the outside world. In cells with nuclei (eukaryotic cells), membranes also form internal compartments (organelles) which carry out specialized tasks, such as protein modification and sorting in the case of the Golgi apparatus, and ATP production in the case of mitochondria. The main components of membranes are lipids and proteins. The proteins can be channels, carriers, receptors, catalysts, signaling molecules, or structural elements, and typically contribute a substantial fraction of the total membrane dry weight. The equilibrium properties of pure lipid membranes are relatively well-understood, and will be the main focus of this article. The framework of elasticity theory and statistical mechanics that we will develop will serve as the foundation for understanding biological phenomena such as the nonequilibrium behavior of membranes laden with ion pumps, the role of membrane elasticity in ion channel gating, and the dynamics of vesicle fission and fusion. Understanding the mechanics of lipid membranes is also important for drug encapsulation and delivery.

Influence of membrane phospholipid composition and structural organization on spontaneous lipid transfer between membranes.

Science.gov (United States)

Pankov, R; Markovska, T; Antonov, P; Ivanova, L; Momchilova, A

2006-09-01

Investigations were carried out on the influence of phospholipid composition of model membranes on the processes of spontaneous lipid transfer between membranes. Acceptor vesicles were prepared from phospholipids extracted from plasma membranes of control and ras-transformed fibroblasts. Acceptor model membranes with manipulated levels of phosphatidylethanolamine (PE), sphingomyelin and phosphatidic acid were also used in the studies. Donor vesicles were prepared of phosphatidylcholine (PC) and contained two fluorescent lipid analogues, NBD-PC and N-Rh-PE, at a self-quenching concentration. Lipid transfer rate was assessed by measuring the increase of fluorescence in acceptor membranes due to transfer of fluorescent lipid analogues from quenched donor to unquenched acceptor vesicles. The results showed that spontaneous NBD-PC transfer increased upon fluidization of acceptor vesicles. In addition, elevation of PE concentration in model membranes was also accompanied by an increase of lipid transfer to all series of acceptor vesicles. The results are discussed with respect to the role of lipid composition and structural order of cellular plasma membranes in the processes of spontaneous lipid exchange between membrane bilayers.

Plasma treatment of polyethersulfone membrane for benzene removal from water by air gap membrane distillation.

Science.gov (United States)

Pedram, Sara; Mortaheb, Hamid Reza; Arefi-Khonsari, Farzaneh

2018-01-01

In order to obtain a durable cost-effective membrane for membrane distillation (MD) process, flat sheet polyethersulfone (PES) membranes were modified by an atmospheric pressure nonequilibrium plasma generated using a dielectric barrier discharge in a mixture of argon and hexamethyldisiloxane as the organosilicon precursor. The surface properties of the plasma-modified membranes were characterized by water contact angle (CA), liquid entry pressure, X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. The water CA of the membrane was increased from 64° to 104° by depositing a Si(CH 3 )-rich thin layer. While the pristine PES membrane was not applicable in the MD process, the modified PES membrane could be applied for the first time in an air gap membrane distillation setup for the removal of benzene as a volatile organic compound from water. The experimental design using central composite design and response surface methodology was applied to study the effects of feed temperature, concentration, and flow rate as well as their binary interactions on the overall permeate flux and separation factor. The separation factor and permeation flux of the modified PES membrane at optimum conditions were comparable with those of commercial polytetrafluoroethylene membrane.

Performance enhancement of membrane electrode assemblies with plasma etched polymer electrolyte membrane in PEM fuel cell

Energy Technology Data Exchange (ETDEWEB)

Cho, Yong-Hun; Yoon, Won-Sub [School of Advanced Materials Engineering, Kookmin University, 861-1 Jeongneung-dong, Seongbuk-gu, Seoul 136-702 (Korea); Bae, Jin Woo; Cho, Yoon-Hwan; Lim, Ju Wan; Ahn, Minjeh; Jho, Jae Young; Sung, Yung-Eun [World Class University (WCU) program of Chemical Convergence for Energy and Environment (C2E2), School of Chemical and Biological Engineering, College of Engineering, Seoul National University (SNU), 599 Gwanak-Ro, Gwanak-gu, Seoul 151-744 (Korea); Kwon, Nak-Hyun [Fuel Cell Vehicle Team 3, Advanced Technology Center, Corporate Research and Development Division, Hyundai-Kia Motors, 104 Mabuk-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-912 (Korea)

2010-10-15

In this work, a surface modified Nafion 212 membrane was fabricated by plasma etching in order to enhance the performance of a membrane electrode assembly (MEA) in a polymer electrolyte membrane fuel cell. Single-cell performance of MEA at 0.7 V was increased by about 19% with membrane that was etched for 10 min compared to that with untreated Nafion 212 membrane. The MEA with membrane etched for 20 min exhibited a current density of 1700 mA cm{sup -2} at 0.35 V, which was 8% higher than that of MEA with untreated membrane (1580 mA cm{sup -2}). The performances of MEAs containing etched membranes were affected by complex factors such as the thickness and surface morphology of the membrane related to etching time. The structural changes and electrochemical properties of the MEAs with etched membranes were characterized by field emission scanning electron microscopy, Fourier transform-infrared spectrometry, electrochemical impedance spectroscopy, and cyclic voltammetry. (author)

Development of highly porous flat sheet polyvinylidene fluoride (PVDF) membranes for membrane distillation

KAUST Repository

Alsaery, Salim A.

2017-05-01

With the increase of population every year, fresh water scarcity has rapidly increased and it is reaching a risky level, particularly in Africa and the Middle East. Desalination of seawater is an essential process for fresh water generation. One of the methods for desalination is membrane distillation (MD). MD process separates an aqueous liquid feed across a porous hydrophobic membrane to produce pure water via evaporation. Polyvinlidene fluoride (PVDF) membranes reinforced with a polyester fabric were fabricated as potential candidates for MD. Non-solvent induced phase separation coupled with steam treatment was used to prepare the PVDF membranes. A portion of the prepared membrane was coated with Teflon (AF2400) to increase its hydrophobicity. In the first study, the fabricated membranes were characterized using scanning electron microscopy and other techniques, and they were evaluated using direct contact MD (DCMD). The fabricated membranes showed a porous sponge-like structure with some macrovoids. The macrovoid formation and the spongy structure in the membrane cross-sections contributed significantly to a high permeate flux as they provide a large space for vapor water transport. The modified PVDF membranes with steaming and coating exhibited a permeate flux of around 40 L/h m2 (i.e. 27-30% increase to the control PVDF membrane) at temperatures of 60 °C (feed) and 20 °C (permeate). This increase in the permeate flux for the modified membranes was mainly attributed to its larger pore size on the bottom surface. In the second study, the control PVDF membrane was tested in two different module designs (i.e. semi-circular pipe and rectangular duct module designs). The semi-circular module design (turbulent regime) exhibited a higher permeate flux, 3-fold higher than that of the rectangular duct module design (laminar regime) at feed temperature of 60 °C. Furthermore, a heat energy balance was performed for each module design to determine the temperature

High flux and antifouling properties of negatively charged membrane for dyeing wastewater treatment by membrane distillation

KAUST Repository

An, Alicia Kyoungjin

2016-07-25

This study investigated the applicability of membrane distillation (MD) to treat dyeing wastewater discharged by the textile industry. Four different dyes containing methylene blue (MB), crystal violet (CV), acid red 18 (AR), and acid yellow 36 (AY) were tested. Two types of hydrophobic membranes made of polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) were used. The membranes were characterized by testing against each dye (foulant-foulant) and the membrane–dye (membrane-foulant) interfacial interactions and their mechanisms were identified. The MD membranes possessed negative charges, which facilitated the treatment of acid and azo dyes of the same charge and showed higher fluxes. In addition, PTFE membrane reduced the wettability with higher hydrophobicity of the membrane surface. The PTFE membrane evidenced especially its resistant to dye absorption, as its strong negative charge and chemical structure caused a flake-like (loose) dye–dye structure to form on the membrane surface rather than in the membrane pores. This also enabled the recovery of flux and membrane properties by water flushing (WF), thereby direct-contact MD with PTFE membrane treating 100 mg/L of dye mixtures showed stable flux and superior color removal during five days operation. Thus, MD shows a potential for stable long-term operation in conjunction with a simple membrane cleaning process, and its suitability in dyeing wastewater treatment.

Amine-functionalized PVA-co-PE nanofibrous membrane as affinity membrane with high adsorption capacity for bilirubin.

Science.gov (United States)

Wang, Wenwen; Zhang, Hao; Zhang, Zhifeng; Luo, Mengying; Wang, Yuedan; Liu, Qiongzhen; Chen, Yuanli; Li, Mufang; Wang, Dong

2017-02-01

In this study, poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membrane was activated by sodium hydroxide and cyanuric chloride, and then the activated membranes were functionalized by 1,3-propanediamine, hexamethylenediamine and diethylenetriamine to be affinity membranes for bilirubin removal, respectively. The chemical structures and morphologies of membranes were investigated by SEM, FTIR and XPS. And the adsorption ability of different amine-functionalized nanofibrous membranes for bilirubin was characterized. Furthermore, the effects of temperature, initial concentration of bilirubin, NaCl concentration and BSA concentration on the adsorption capacity for bilirubin of diethylenetriamine-functionalized nanofibrous membrane were studied. Results indicated that the adsorption capacity for bilirubin of diethylenetriamine-functionalized nanofibrous membrane could reach 85mg/g membrane when the initial bilirubin concentration was 200mg/L while the adsorption capacity could be increased to 110mg/g membrane if the initial bilirubin concentration was more than 400mg/L. The dynamic adsorption of diethylenetriamine-functionalized nanofibrous membrane showed that the ligands of amine groups on the membrane surface could be used as far as possible by recirculating the plasma with certain flow rates. Therefore, the diethylenetriamine-functionalized PVA-co-PE nanofibrous membrane possessed high adsorption capacity for bilirubin and it can be candidate as affinity membrane for bilirubin removal. Copyright © 2016. Published by Elsevier B.V.

Using biomass of starch-rich transgenic Arabidopsis vacuolar as feedstock for fermentative hydrogen production

Energy Technology Data Exchange (ETDEWEB)

Lo, Yung-Chung; Cheng, Chieh-Lun; Chen, Chun-Yen [National Cheng Kung Univ., Tainan, Taiwan (China). Dept. of Chemical Engineering; Huang, Li-Fen; Chang, Jo-Shu [Yuan Ze Univ., Tao-yuan, Taiwan (China). Graduate School of Biotechnology and Bioengineering

2010-07-01

Cellulose is the major constitute of plant biomass and highly available in agricultural wastes and industrial effluents, thereby being a cost-effective feedstock for bioenergy production. However, most hydrogen producing bacteria (HPB) could not directly convert cellulosic materials (such as rice husk and rice straw) into hydrogen whereas most HPB could utilize sugar and starch for hydrogen production. In this work, we used an indigenous bacterial isolate Clostridium butyricum CGS2 as HPB, which could directly convert soluble starch into H2 with a maximum H2 production rate and a H2 yield of 205.07 ml H2/h/l and 6.46 mmol H2/g starch, respectively. However, C. butyricum CGS2 could not ferment pure cellulosic materials such as carboxymethyl cellulose and xylan. Moreover, we found that C. butyricum CGS2 could utilize rich husk to produce H2 at a rate of 13.19 ml H2/h/l due to the starch content in rice husk (H2 yield = 1.49 mmol H2/g rice husk). In contrast, since lacking starch content, rice straw cannot be converted to H2 by C. butyricum CGS2. The foregoing results suggest that increasing the starch content in the natural agricultural wastes may make them better feedstock for fermentative H2 production. Hence, a genetically modified plant (Arabidopsis vacuolar) was constructed to enhance its starch concentration. The starch concentration of mutant plant S1 increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using mutant plant S1 as carbon source, C. butyricum CGS2 was able to give a high cumulative H2 production and H2 production rate of 285.4 ml H2/l and 43.6 ml/h/l, respectively. The cumulative H2 production and H2 production rate both increased when the concentration of the transgenic plant was increased. Therefore, this study successful demonstrated the feasibility of expressing starch on genetically-modified plants to create a more effective feedstock for dark H2 fermentation. (orig.)

Hydrogen Selective Exfoliated Zeolite Membranes

Energy Technology Data Exchange (ETDEWEB)

Tsapatsis, Michael [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Daoutidis, Prodromos [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Elyassi, Bahman [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Lima, Fernando [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Iyer, Aparna [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Agrawal, Kumar [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Sabnis, Sanket [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science

2015-04-06

The objective of this project was to develop and evaluate an innovative membrane technology at process conditions that would be representative of Integrated Gasification Combined Cycle (IGCC) advanced power generation with pre-combustion capture of carbon dioxide (CO₂). This research focused on hydrogen (H₂)-selective zeolite membranes that could be utilized to separate conditioned syngas into H₂-rich and CO₂-rich components. Both experiments and process design and optimization calculations were performed to evaluate the concept of ultra-thin membranes made from zeolites nanosheets. In this work, efforts in the laboratory were made to tackle two fundamental challenges in application of zeolite membranes in harsh industrial environments, namely, membrane thickness and membrane stability. Conventional zeolite membranes have thicknesses in the micron range, limiting their performance. In this research, we developed a method for fabrication of ultimately thin zeolite membranes based on zeolite nanosheets. A range of layered zeolites (MWW, RWR, NSI structure types) suitable for hydrogen separation was successfully exfoliated to their constituent nanosheets. Further, membranes were made from one of these zeolites, MWW, to demonstrate the potential of this group of materials. Moreover, long-term steam stability of these zeolites (up to 6 months) was investigated in high concentrations of steam (35 mol% and 95 mole%), high pressure (10 barg), and high temperatures (350 °C and 600 °C) relevant to conditions of water-gas-shift and steam methane reforming reactions. It was found that certain nanosheets are stable, and that stability depends on the concentration of structural defects. Additionally, models that represent a water-gas-shift (WGS) membrane reactor equipped with the zeolite membrane were developed for systems studies. These studies had the aim of analyzing the effect of the membrane reactor integration into IGCC plants

Bacterial membrane proteomics.

Science.gov (United States)

Poetsch, Ansgar; Wolters, Dirk

2008-10-01

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

Biocatalytic Self-Cleaning Polymer Membranes

Directory of Open Access Journals (Sweden)

Agnes Schulze

2015-09-01

Full Text Available Polymer membrane surfaces have been equipped with the digestive enzyme trypsin. Enzyme immobilization was performed by electron beam irradiation in aqueous media within a one-step method. Using this method, trypsin was covalently and side-unspecific attached to the membrane surface. Thus, the use of preceding polymer functionalization and the use of toxic solvents or reagents can be avoided. The resulting membranes showed significantly improved antifouling properties as demonstrated by repeated filtration of protein solutions. Furthermore, the biocatalytic membrane can be simply “switched on” to actively degrade a fouling layer on the membrane surface and regain the initial permeability. The membrane pore structure (pore size and porosity was neither damaged by the electron beam treatment nor blocked by the enzyme loading, ensuring a stable membrane performance.

RADIATION STABILITY OF NAFION MEMBRANES USED FOR ISOTOPE SEPARATION BY PROTON EXCHANGE MEMBRANE ELECTROLYSIS

International Nuclear Information System (INIS)

Fox, E.

2009-01-01

Proton Exchange Membrane Electrolyzers have potential interest for use for hydrogen isotope separation from water. In order for PEME to be fully utilized, more information is needed on the stability of Nafion when exposed to radiation. This work examines Nafion 117 under varying exposure conditions, including dose rate, total dosage and atmospheric condition. Analytical tools, such as FT-IR, ion exchange capacity, DMA and TIC-TOC were used to characterize the exposed membranes. Analysis of the water from saturated membranes can provide important data on the stability of the membranes during radiation exposure. It was found that the dose rate of exposure plays an important role in membrane degradation. Potential mechanisms for membrane degradation include peroxide formation by free radicals

RADIATION STABILITY OF NAFION MEMBRANES USED FOR ISOTOPE SEPARATION BY PROTON EXCHANGE MEMBRANE ELECTROLYSIS

Energy Technology Data Exchange (ETDEWEB)

Fox, E

2009-05-15

Proton Exchange Membrane Electrolyzers have potential interest for use for hydrogen isotope separation from water. In order for PEME to be fully utilized, more information is needed on the stability of Nafion when exposed to radiation. This work examines Nafion 117 under varying exposure conditions, including dose rate, total dosage and atmospheric condition. Analytical tools, such as FT-IR, ion exchange capacity, DMA and TIC-TOC were used to characterize the exposed membranes. Analysis of the water from saturated membranes can provide important data on the stability of the membranes during radiation exposure. It was found that the dose rate of exposure plays an important role in membrane degradation. Potential mechanisms for membrane degradation include peroxide formation by free radicals.

Dicty_cDB: [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available VF (Link to library) VFB804 (Link to dictyBase) - - - Contig-U13849-1 VFB804P (Link... to Original site) VFB804F 150 VFB804Z 637 VFB804P 787 - - Show VFB804 Library VF (Link to library) Clone ID VFB804 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U13849-1 Original site URL http://dict... nuclear 4.0 %: cytoskeletal 4.0 %: vacuolar 4.0 %: plasma membrane 4.0 %: vesicles of secretory system >> predict

Review on Development of Ceramic Membrane From Sol-Gel Route: Parameters Affecting Characteristics of the Membrane

Directory of Open Access Journals (Sweden)

M. R. Othman and H. Mukhtar

2012-08-01

Full Text Available The importance of laboratory scale ceramic membrane preparation using sol-gel technique with pore sizes in the range of 1-10nm is reviewed. Parameters affecting the characteristics of membrane during membrane development are highlighted and discussed in detail. Experimental results from literatures have shown that the correct amount of acid, water, PVA, appropriate membrane thickness, proper control of drying rate, and appropriate temperature profile selection during sintering process are necessary in order to acquire sufficient strength and reduce the formation of crack in the membrane. The different temperature setting during sintering process also influences the size of pore formed.Key Words: Sol-Gel, Inorganic Membrane, Ceramic Membrane, Gas Permeation, Sintering, Sol Properties, Membrane Morphologies, Pore Size Distribution.

Polymeric molecular sieve membranes via in situ cross-linking of non-porous polymer membrane templates.

Science.gov (United States)

Qiao, Zhen-An; Chai, Song-Hai; Nelson, Kimberly; Bi, Zhonghe; Chen, Jihua; Mahurin, Shannon M; Zhu, Xiang; Dai, Sheng

2014-04-16

High-performance polymeric membranes for gas separation are attractive for molecular-level separations in industrial-scale chemical, energy and environmental processes. Molecular sieving materials are widely regarded as the next-generation membranes to simultaneously achieve high permeability and selectivity. However, most polymeric molecular sieve membranes are based on a few solution-processable polymers such as polymers of intrinsic microporosity. Here we report an in situ cross-linking strategy for the preparation of polymeric molecular sieve membranes with hierarchical and tailorable porosity. These membranes demonstrate exceptional performance as molecular sieves with high gas permeabilities and selectivities for smaller gas molecules, such as carbon dioxide and oxygen, over larger molecules such as nitrogen. Hence, these membranes have potential for large-scale gas separations of commercial and environmental relevance. Moreover, this strategy could provide a possible alternative to 'classical' methods for the preparation of porous membranes and, in some cases, the only viable synthetic route towards certain membranes.

Polybenzimidazole/Mxene composite membranes for intermediate temperature polymer electrolyte membrane fuel cells

Science.gov (United States)

Fei, Mingming; Lin, Ruizhi; Deng, Yuming; Xian, Hongxi; Bian, Renji; Zhang, Xiaole; Cheng, Jigui; Xu, Chenxi; Cai, Dongyu

2018-01-01

This report demonstrated the first study on the use of a new 2D nanomaterial (Mxene) for enhancing membrane performance of intermediate temperature (>100 °C) polymer electrolyte membrane fuel cells (ITPEMFCs). In this study, a typical Ti3C2T x -MXene was synthesized and incorporated into polybenzimidazole (PBI)-based membranes by using a solution blending method. The composite membrane with 3 wt% Ti3C2T x -MXene showed the proton conductivity more than 2 times higher than that of pristine PBI membrane at the temperature range of 100 °C-170 °C, and led to substantial increase in maximum power density of fuel cells by ˜30% tested at 150 °C. The addition of Ti3C2T x -MXene also improved the mechanical properties and thermal stability of PBI membranes. At 3 wt% Ti3C2T x -MXene, the elongation at break of phosphoric acid doped PBI remained unaffected at 150 °C, and the tensile strength and Young’s modulus was increased by ˜150% and ˜160%, respectively. This study pointed out promising application of MXene in ITPEMFCs.

Microfabricated hydrogen sensitive membranes

Energy Technology Data Exchange (ETDEWEB)

Naddaf, A.; Kraetz, L. [Lehrstuhl fuer Thermische Verfahrenstechnik, Technische Universitaet Kaiserslautern (Germany); Detemple, P.; Schmitt, S.; Hessel, V. [Institut fuer Mikrotechnik Mainz GmbH, Mainz (Germany); Faqir, N. [University of Jordan, Amman (Jordan); Bart, H.J.

2009-01-15

Thin, defect-free palladium, palladium/copper and palladium/silver hydrogen absorbing membranes were microfabricated. A dual sputtering technique was used to deposit the palladium alloy membranes of only 1 {mu}m thickness on a nonporous silicon substrate. Advanced silicon etching (ASE) was applied on the backside to create a mechanically stable support structure for the thin films. Performance evaluation was carried out for different gases in a temperature range of 20 C to 298 C at a constant differential pressure of 110 kPa at the two sides of the membrane. The composite membranes show an excellent permeation rate of hydrogen, which appears to be 0.05 Pa m{sup 3} s{sup -1} and 0.01.10{sup -3} Pa m{sup 3} s{sup -1} at 20 C for the microfabricated 23 % silver and the 53 % copper composite membranes, respectively. The selectivity to hydrogen over a gas mixture containing, in addition to hydrogen, carbon monoxide, carbon dioxide and nitrogen was measured. The mass spectrometer did not detect any CO{sub 2} or CO, showing that the membrane is completely hydrogen selective. The microfabricated membranes exhibit both high mechanical strength (they easily withstand pressures up to 4 bar) and high thermal stability (up to 650 C). (Abstract Copyright [2009], Wiley Periodicals, Inc.)

Characterising antimicrobial protein-membrane complexes

International Nuclear Information System (INIS)

Xun, Gloria; Dingley, Andrew; Tremouilhac, Pierre

2009-01-01

Full text: Antimicrobial proteins (AMPs) are host defence molecules that protect organisms from microbial infection. A number of hypotheses for AMP activity have been proposed which involve protein membrane interactions. However, there is a paucity of information describing AMP-membrane complexes in detail. The aim of this project is to characterise the interactions of amoebapore-A (APA-1) with membrane models using primarily solution-state NMR spectroscopy. APA-1 is an AMP which is regulated by a pH-dependent dimerisation event. Based on the atomic resolution solution structure of monomeric APA-1, it is proposed that this dimerisation is a prerequisite for ring-like hexameric pore formation. Due to the cytotoxicity of APA-1, we have developed a cell-free system to produce this protein. To facilitate our studies, we have adapted the cell-free system to isotope label APA-1. 13 C /15 N -enriched APA-1 sample was achieved and we have begun characterising APA-1 dimerisation and membrane interactions using NMR spectroscopy and other biochemical/biophysical methods. Neutron reflectometry is a surface-sensitive technique and therefore represents an ideal technique to probe how APA-1 interacts with membranes at the molecular level under different physiological conditions. Using Platypus, the pH-induced APA-1-membrane interactions should be detectable as an increase of the amount of protein adsorbed at the membrane surface and changes in the membrane properties. Specifically, detailed information of the structure and dimensions of the protein-membrane complex, the position and amount of the protein in the membrane, and the perturbation of the membrane phospholipids on protein incorporation can be extracted from the neutron reflectometry measurement. Such information will enable critical assessment of current proposed mechanisms of AMP activity in bacterial membranes and complement our NMR studies