Toxoplasma gondii GRA7-Targeted ASC and PLD1 Promote Antibacterial Host Defense via PKCα.

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Hyun-Jung Koh

2017-01-01

Full Text Available Tuberculosis is a global health problem and at least one-third of the world's population is infected with Mycobacterium tuberculosis (MTB. MTB is a successful pathogen that enhances its own intracellular survival by inhibiting inflammation and arresting phago-lysosomal fusion. We previously demonstrated that Toxoplasma gondii (T. gondii dense granule antigen (GRA 7 interacts with TNF receptor-associated factor 6 via Myeloid differentiation primary response gene 88, enabling innate immune responses in macrophages. To extend these studies, we found that GRA7 interacts with host proteins involved in antimicrobial host defense mechanisms as a therapeutic strategy for tuberculosis. Here, we show that protein kinase C (PKCα-mediated phosphorylation of T. gondii GRA7-I (Ser52 regulates the interaction of GRA7 with PYD domain of apoptosis-associated speck-like protein containing a carboxy-terminal CARD, which is capable of oligomerization and inflammasome activation can lead to antimicrobial defense against MTB. Furthermore, GRA7-III interacted with the PX domain of phospholipase D1, facilitating its enzyme activity, phago-lysosomal maturation, and subsequent antimicrobial activity in a GRA7-III (Ser135 phosphorylation-dependent manner via PKCα. Taken together, these results underscore a previously unrecognized role of GRA7 in modulating antimicrobial host defense mechanism during mycobacterial infection.

Stavovi hrvatskih građana prema kulturnim različitostima

OpenAIRE

Mesić, Milan; Bagić, Dragan

2011-01-01

Članak se temelji na dijelu rezultata reprezentativnoga nacionalnog ispitivanja stavova hrvatskih građana o kulturnim različitostima u hrvatskome društvu. Terensko anketiranje provedeno je metodom osobnog intervjua u kućanstvu ispitanika, u okviru omnibus istraživanja. Pod kulturnim različitostima autori misle na nacionalne manjine i vjerske zajednice. U tom smislu Hrvatska je, poput većine zemalja suvremenoga svijeta, kulturno heterogena politička zajednica. Stoga će odnos njezinih građana p...

The plant vacuolar Na+/H+ antiport.

Science.gov (United States)

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 respiration of nitrate coupled to energy conservation in filamentous Beggiatoaceae.

Science.gov (United States)

Beutler, Martin; Milucka, Jana; Hinck, Susanne; Schreiber, Frank; Brock, Jörg; Mussmann, Marc; Schulz-Vogt, Heide N; de Beer, Dirk

2012-11-01

We show that the nitrate storing vacuole of the sulfide-oxidizing bacterium Candidatus Allobeggiatoa halophila has an electron transport chain (ETC), which generates a proton motive force (PMF) used for cellular energy conservation. Immunostaining by antibodies showed that cytochrome c oxidase, an ETC protein and a vacuolar ATPase are present in the vacuolar membrane and cytochrome c in the vacuolar lumen. The effect of different inhibitors on the vacuolar pH was studied by pH imaging. Inhibition of vacuolar ATPases and pyrophosphatases resulted in a pH decrease in the vacuole, showing that the proton gradient over the vacuolar membrane is used for ATP and pyrophosphate generation. Blockage of the ETC decreased the vacuolar PMF, indicating that the proton gradient is build up by an ETC. Furthermore, addition of nitrate resulted in an increase of the vacuolar PMF. Inhibition of nitrate reduction, led to a decreased PMF. Nitric oxide was detected in vacuoles of cells exposed to nitrate showing that nitrite, the product of nitrate reduction, is reduced inside the vacuole. These findings show consistently that nitrate respiration contributes to the high proton concentration within the vacuole and the PMF over the vacuolar membrane is actively used for energy conservation. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Lipid nanotechnologies for structural studies of membrane-associated proteins.

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Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

2014-11-01

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

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.

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.

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

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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(+)

Characterization of a membrane-associated serine protease in Escherichia coli

International Nuclear Information System (INIS)

Palmer, S.M.; St John, A.C.

1987-01-01

Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [ 3 H]diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin

GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM.

Science.gov (United States)

Hauer, Florian; Gerle, Christoph; Fischer, Niels; Oshima, Atsunori; Shinzawa-Itoh, Kyoko; Shimada, Satoru; Yokoyama, Ken; Fujiyoshi, Yoshinori; Stark, Holger

2015-09-01

We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

Graça square in Salvador – BA: a “new” perception Largo da Graça em Salvador-BA: Uma “nova” percepção

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Denise Silva Magalhães

2012-07-01

Full Text Available The experience undertaken along an empirical work at Largo da Graça (Graça Square, in Salvador, Bahia, motivated this article which presents a deep study with new researches, local visits, interviews and pictures, as a reality direct observation technique. But, which method to adopt in order to learn the essence of the studied space? Following a thought by Júlia Adão Bernardes, I strayed without a trail, expecting to have trailed it at the end of the work. The activities were systematized and rationalized in order to lead to a valid knowledge of the area; I proceeded the investigations and analysis of the facts, processes and institutions of the past, which interacted with the qualitative interviews on the local workers, inhabitants and visitors everyday life. My perception of the Largo da Graça has changed from a simply perceived to a lived space, a differentiated public space, through its concreteness and the daily practices and social dynamics there occurring.Experiência vivenciada durante um pequeno trabalho empírico realizado no Largo da Graça, em Salvador-BA, motivou este artigo que apresenta o estudo aprofundado com novas pesquisas, várias visitas ao local, entrevistas e fotos, como técnicas de observação direta da realidade. Mas, qual método deveria seguir para apreender a essência do espaço em questão? Seguindo uma ponderação de Júlia Adão Bernardes, caminhei sem caminho, esperando tê-lo feito ao final do trabalho. Sistematizei e racionalizei as atividades, para obter um conhecimento válido da área; procedi a investigações e análises de fatos, processos e instituições do passado, que interagiram com entrevistas qualitativas sobre o cotidiano dos frequentadores, moradores e trabalhadores do local. Transformou-se minha percepção do Largo da Graça, de um espaço apenas percebido, para um espaço vivido, um espaço público diferenciado por sua concretude e por práticas cotidianas e dinâmicas sociais que

Localization of membrane-associated calcium in unpollinated and pollinated pistil of Petunia hybrida Hort.

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Elżbieta Bednarska

2014-01-01

Full Text Available In the pistil of Petunia hybrida, the transmitting tract and the ovules are the sites which give Ca2+-CTC fluorescence. In unpollinated pistil the level of membrane-associated Ca2+ decreases from the stigma to the base of the style. The renewed strong rise of Ca2+-CTC fluorescence appears on the placenta surface and in the ovule integuments. Following pollination, when the pollen tubes have grown through the pistil, the pattern of membrane-associated Ca2+ on the path stigma - ovary is reversed. The highest fluorescence is found in the base of the style. In pollinated ovules the Ca2+-CTC fluorescence increases markedly. In the transmitting cells membrane-associated Ca2+ occurs mainly in the polar regions of the cell. During cell degeneration following pollination the cytoplasmic clusters show Ca2+-CTC fluorescence. The used P. hybrida cultivar is self-fertile. The selection of pollen tubes occurs mainly in the upper part of the style. The rejected pollen tubes show a steady high level of membrane-associated calcium.

Genetic Diversity of Toxoplasma gondii Strains from Different Hosts and Geographical Regions by Sequence Analysis of GRA20 Gene.

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Ning, Hong-Rui; Huang, Si-Yang; Wang, Jin-Lei; Xu, Qian-Ming; Zhu, Xing-Quan

2015-06-01

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.

Applying GRA and QFD to Improve Library Service Quality

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Chen, Yen-Ting; Chou, Tsung-Yu

2011-01-01

This paper applied Grey Relational Analysis (GRA) to Quality Function Deployment (QFD) to identify service improvement techniques for an academic library. First, reader needs and their importance, and satisfaction degrees were examined via questionnaires. Second, the service improvement techniques for satisfying the reader needs were developed by…

Evolutionarily conserved 5'-3' exoribonuclease Xrn1 accumulates at plasma membrane-associated eisosomes in post-diauxic yeast.

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Tomas Grousl

Full Text Available Regulation of gene expression on the level of translation and mRNA turnover is widely conserved evolutionarily. We have found that the main mRNA decay enzyme, exoribonuclease Xrn1, accumulates at the plasma membrane-associated eisosomes after glucose exhaustion in a culture of the yeast S. cerevisiae. Eisosomal localization of Xrn1 is not achieved in cells lacking the main component of eisosomes, Pil1, or Sur7, the protein accumulating at the membrane compartment of Can1 (MCC - the eisosome-organized plasma membrane microdomain. In contrast to the conditions of diauxic shift, when Xrn1 accumulates in processing bodies (P-bodies, or acute heat stress, in which these cytosolic accumulations of Xrn1 associate with eIF3a/Rpg1-containing stress granules, Xrn1 is not accompanied by other mRNA-decay machinery components when it accumulates at eisosomes in post-diauxic cells. It is important that Xrn1 is released from eisosomes after addition of fermentable substrate. We suggest that this spatial segregation of Xrn1 from the rest of the mRNA-decay machinery reflects a general regulatory mechanism, in which the key enzyme is kept separate from the rest of mRNA decay factors in resting cells but ready for immediate use when fermentable nutrients emerge and appropriate metabolism reprogramming is required. In particular, the localization of Xrn1 to the eisosome, together with previously published data, accents the relevance of this plasma membrane-associated compartment as a multipotent regulatory site.

The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action.

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Chaili, Siyang; Cheung, Ambrose L; Bayer, Arnold S; Xiong, Yan Q; Waring, Alan J; Memmi, Guido; Donegan, Niles; Yang, Soo-Jin; Yeaman, Michael R

2016-02-01

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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.

Immunization of C57BL/6 Mice with GRA2 Combined with MPL Conferred Partial Immune Protection against Toxoplasma gondii

Science.gov (United States)

Babaie, Jalal; Amiri, Samira; Homayoun, Robab; Azimi, Ebrahim; Mohabati, Reyhaneh; Berizi, Mahboobe; Sadaie, M. Reza; Golkar, Majid

2018-01-01

We have previously reported that immunization with GRA2 antigen of Toxoplasma gondii induces protective immunity in CBA/J (H2k) and BALB/c mice (H2d). We aimed to examine whether immunization of a distinct strain of rodent with recombinant dense granule antigens (GRA2) combined with monophosphorryl lipid A (MPL) adjuvant elicits protective immune response against T. gondii. C57BL/6 (H2b haplotype) mice were immunized with GRA2, formulated in MPL adjuvant. Strong humoral response, predominantly of IgG1 subclass and cellular response, IFN-γ, was detected at three weeks post immunization. Mice immunized with GRA2 had significantly (p < 0.01) fewer brain cysts than those in the adjuvant group, upon challenge infection. Despite the production of a strong antibody response, IFN-γ production and brain cyst reduction were not significant when the immunized mice were infected four months after the immunization. We can conclude that GRA2 immunization partially protects against T. gondii infection in C57BL/6 mice, though the potency and longevity of this antigen as a standalone vaccine may vary in distinct genetic backgrounds. This observation further emphasizes the utility of GRA2 for incorporation into a multi-antigenic vaccine against T. gondii.

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.

Immunodiagnosis and molecular validation of Toxoplasma gondii-recombinant dense granular (GRA) 7 protein for the detection of toxoplasmosis in patients with cancer.

Science.gov (United States)

Arab-Mazar, Zahra; Fallahi, Shirzad; Koochaki, Ameneh; Haghighi, Ali; Seyyed Tabaei, Seyyed Javad

2016-02-01

Serological assays for the diagnosis of toxoplasmosis mostly rely on the tachyzoite specific antigens of Toxoplasma gondii, which are difficult to produce by conventional methods. The aim of this study was to clone and express of GRA7 protein of T. gondii and evaluate its potential for immunodiagnosis of toxoplasmosis in cancer patients. As well as validate the results using a new molecular assay, LAMP technique. The GRA7 gene was successfully cloned, expressed and purified by affinity chromatography and the production was evaluated by SDS PAGE, dot blot and western blot analyses. The rGRA7 was used for developing an ELISA based on the rGRA7 using sera from patients with toxoplasmosis and healthy controls. Furthermore, 50 serum samples from leukemic children infected with toxoplasmosis and 50 seronegative controls were included to evaluate the sensitivity and specificity of rGRA7 based ELISA. Finally, the LAMP technique was used to assess the accuracy and validity of the results obtained by rGRA7 based ELISA. The consistency of the results of two tests was determined by using the Kappa coefficient of agreement. The rGRA7 showed higher and optimum immunoreactivity with 1:100 dilution of serum from Toxoplasma infected patients. The sensitivity and specificity of test were calculated as 92 and 94%, respectively. According to the Kappa coefficient of agreement, there was a significant conformance between the results obtained by ELISA based on the rGRA7 and the results of LAMP technique (≈96%, Ptoxoplasmosis in patients including patients with cancer. Copyright © 2015. Published by Elsevier GmbH.

VisGraB: A Benchmark for Vision-Based Grasping. Paladyn Journal of Behavioral Robotics

DEFF Research Database (Denmark)

Kootstra, Gert; Popovic, Mila; Jørgensen, Jimmy Alison

2012-01-01

that a large number of grasps can be executed and evaluated while dealing with dynamics and the noise and uncertainty present in the real world images. VisGraB enables a fair comparison among different grasping methods. The user furthermore does not need to deal with robot hardware, focusing on the vision......We present a database and a software tool, VisGraB, for benchmarking of methods for vision-based grasping of unknown objects with no prior object knowledge. The benchmark is a combined real-world and simulated experimental setup. Stereo images of real scenes containing several objects in different...

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.

siRNA-based Analysis of the Abrogation of the Protective Function of Membrane-associated Catalase of Tumor Cells.

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Bauer, Georg

2017-02-01

Tumor cells, in contrast to non-malignant cells, show sustained expression of membrane-associated NADPH oxidase-1 and therefore generate extracellular superoxide anions and their dismutation product H 2 O 2 In order to prevent intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling, tumor cells need to express membrane-associated catalase that interferes with HOCl and nitric oxide/peroxynitrite signaling. Catalase is attached to tumor cells through the activity of transglutaminase-2 and is prevented from superoxide anion-dependent inhibition through coexpression of membrane-associated superoxide dismutase. Therefore, specific inhibition of membrane-associated catalase should reactivate intercellular ROS/RNS-dependent apoptosis-inducing signaling. These processes are analyzed here through small interfering RNA-mediated knockdown of essential signaling compounds. This allows to establish a rather comprehensive picture of intercellular ROS/RNS signaling that may be instrumental for future therapeutic approaches. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

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Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

2008-12-01

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

Science.gov (United States)

Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

2013-04-17

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells 1

Science.gov (United States)

Arora, Rajeev; Palta, Jiwan P.

1988-01-01

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K+, along with small but significant loss of cellular Ca2+. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca2+ from membrane by extracellular K+ and subsequent perturbation of K+ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca2+ compared to control. Loss of membrane-associated Ca2+ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca2+-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl2. Our results suggest that the loss of membrane-associated Ca2+, in part, plays a role in initiation and progression of freezing injury. Images Fig. 1 Fig. 2 PMID:16666196

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.

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.

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

Science.gov (United States)

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.

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

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Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-09-12

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9*

Science.gov (United States)

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-01-01

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al3+ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. PMID:25028514

Fotografski albumi u funkciji građenja popularnog mita

OpenAIRE

Ćalović, Dragan

2014-01-01

U tekstu se analiziraju principi građenja popularnog mita. Popularni mit sagledan je kao rezultat popularne proizvodnje koja eksploatira materiju metajezika kao drugostupanjskog semiološkog sustava. Za razliku od Barthesovog koncepta mitifikacije samog mita, ili popularnog korištenja mita u duhu Fiskeovog razumijevanja snaga popularnog, popularni mit svoj izraz pronalazi u slobodnom korištenju potencijala metajezika. Popularni mit preuzima mehanizme metajezične značenjske produkcije kako bi m...

Largo da Graça em Salvador-BA: Uma “nova” percepção

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Denise Silva Magalhães

2012-07-01

Full Text Available Experiência vivenciada durante um pequeno trabalho empírico realizado no Largo da Graça, em Salvador-BA, motivou este artigo que apresenta o estudo aprofundado com novas pesquisas, várias visitas ao local, entrevistas e fotos, como técnicas de observação direta da realidade. Mas, qual método deveria seguir para apreender a essência do espaço em questão? Seguindo uma ponderação de Júlia Adão Bernardes, caminhei sem caminho, esperando tê-lo feito ao final do trabalho. Sistematizei e racionalizei as atividades, para obter um conhecimento válido da área; procedi a investigações e análises de fatos, processos e instituições do passado, que interagiram com entrevistas qualitativas sobre o cotidiano dos frequentadores, moradores e trabalhadores do local. Transformou-se minha percepção do Largo da Graça, de um espaço apenas percebido, para um espaço vivido, um espaço público diferenciado por sua concretude e por práticas cotidianas e dinâmicas sociais que nele se desenvolvem. Abstract GRAÇA SQUARE IN SALVADOR – BA: A “NEW” PERCEPTION The experience undertaken along an empirical work at Largo da Graça (Graça Square, in Salvador, Bahia, motivated this article which presents a deep study with new researches, local visits, interviews and pictures, as a reality direct observation technique. But, which method to adopt in order to learn the essence of the studied space? Following a thought by Júlia Adão Bernardes, I strayed without a trail, expecting to have trailed it at the end of the work. The activities were systematized and rationalized in order to lead to a valid knowledge of the area; I proceeded the investigations and analysis of the facts, processes and institutions of the past, which interacted with the qualitative interviews on the local workers, inhabitants and visitors everyday life. My perception of the Largo da Graça has changed from a simply perceived to a lived space, a differentiated public space

N=4 scattering amplitudes and the deformed Graßmannian

International Nuclear Information System (INIS)

Ferro, Livia; Łukowski, Tomasz; Staudacher, Matthias

2014-01-01

Some time ago the general tree-level scattering amplitudes of N=4 Super Yang–Mills theory were expressed as certain Graßmannian contour integrals. These remarkable formulas allow to clearly expose the super-conformal, dual super-conformal, and Yangian symmetries of the amplitudes. Using ideas from integrability it was recently shown that the building blocks of the amplitudes permit a natural multi-parameter deformation. However, this approach had been criticized by the observation that it seemed impossible to reassemble the building blocks into Yangian-invariant deformed non-MHV amplitudes. In this note we demonstrate that the deformations may be succinctly summarized by a simple modification of the measure of the Graßmannian integrals, leading to a Yangian-invariant deformation of the general tree-level amplitudes. Interestingly, the deformed building blocks appear as residues of poles in the spectral parameter planes. Given that the contour integrals also contain information on the amplitudes at loop-level, we expect the deformations to be useful there as well. In particular, applying meromorphicity arguments, they may be expected to regulate all notorious infrared divergences. We also point out relations to Gelfand hypergeometric functions and the quantum Knizhnik–Zamolodchikov equations

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

Graphic-Card Cluster for Astrophysics (GraCCA) -- Performance Tests

OpenAIRE

Schive, Hsi-Yu; Chien, Chia-Hung; Wong, Shing-Kwong; Tsai, Yu-Chih; Chiueh, Tzihong

2007-01-01

In this paper, we describe the architecture and performance of the GraCCA system, a Graphic-Card Cluster for Astrophysics simulations. It consists of 16 nodes, with each node equipped with 2 modern graphic cards, the NVIDIA GeForce 8800 GTX. This computing cluster provides a theoretical performance of 16.2 TFLOPS. To demonstrate its performance in astrophysics computation, we have implemented a parallel direct N-body simulation program with shared time-step algorithm in this system. Our syste...

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.

A DYNAMIC APPROACH TO IMPROVING THE EXECUTION OF DEVELOPMENT STRATEGIES FOR DATE-RELATED INDUSTRIES IN IRAN, BASED ON HYBRID GRA AND ISM METHODS

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S. Ghane

2012-01-01

Full Text Available

ENGLISH ABSTRACT: This paper presents a hybrid Grey Relational Analysis (GRA and Interpretive Structural Modelling (ISM methodology for appropriate dynamic analysis, in order to improve the execution of development strategies for date-related industries in Iran. The proposed framework consists of four main phases: (1 establishment of strategies; (2 GRA analysis; (3 binary comparison to determine hierarchy levels using the ISM method; and (4 dynamic analysis. The analysis reveals that the strategic priorities to improve Iran’s date industries can be arranged on three levels. The first level consists of the by-products of date packing and processing. On the second level, three tactics are determined: developing ready-for-use date products, deriving date fruit products, and fermentation of products. The third level is to blend dates with other foods and palm products (excluding dates.

AFRIKAANSE OPSOMMING: Hierdie artikel hou ’n hibirede GRA en ISM metodologie voor vir dinamiese analise om ont-wikkelingstrategieë vir die dadelindustrie in Iran te verbeter. Die voorgestelde raamwerk bestaan uit vier fases: (1 vasstelling van die strategieë; (2 GRA analise; (3 binêre vergelyking om die hierargievlakke te bepaal deur die ISM metode te gebruik; en (4 dinamiese analise. Die analise toon dat die strategiese prioriteite om die dadelindustrie in Iran te verbeter in drie vlakke gerangskik kan word. Die eerste vlak behels die neweprodukte van dadelverpakking en -prosessering. Op die tweede vlak word drie taktieke ontwikkel: die ontwikkeling van gereed-vir-gebruik dadelprodukte, dadelvrugteprodukte en die fermentasie van produkte. Die derde vlak behels die versny van dadels met ander voedsel- en palmprodukte (uitgesonderd dadels.

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.

AN EVALUATION STUDY OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA USING RECOMBINANT PROTEIN GRA1 FOR DETECTION OF IGG ANTIBODIES AGAINTS TOXOPLASMA GONDII INFECTIONS

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Nina Difla Muflikhah

2017-08-01

Full Text Available Toxoplasmosis is an infectious disease caused by Toxoplasma gondii, an intracellular protozoan parasite that live inside the cells of the reticulo endothelial and parenchymal cells of human and animals (mammals and birds. Some cases of toxoplasmosis usually have no symptoms, but in any cases caused severe symptoms, such as hydrocephalus, microcephalus, intracranial calcification, retinal damage, brain abscess, mental retardation, lymphadenopathy, and others. Its severe symptoms usually showed a long time after first exposure, except symptoms showed by congenital transmission caused by infected mother. Early diagnosis is important to prevent the illness but methods for toxoplasmosis screening are still too expensive for developing country. Enzyme-linked immunosorbent assay (ELISA allow the testing of a large number samples within short time frame and based on antibody or antigen detection. This study aimed to know the sensitivity and specificity of recombinat protein GRA1 as antigen using ELISA methods. We tested the sensitivity and spesificity of GRA1 protein as antigen in ELISA methods to diagnose toxoplasmosis and compared with ELISA Kit Commercial. Reliable laboratory testing is important to detect Toxoplasma gondii infection, and focused to improving the low cost and easy-to-use diagnostic instrument. Seventy sera collected and tested using both indirect ELISA, commercial ELISA kit and GRA1 protein coated as antigen. Fourty eight and fifty one samples showed positive IgG antibody result of ELISA-GRA1 and ELISA kit. Negative sample tested by ELISA-GRA1 was 22 samples and 19 sample tested by ELISA Kit. The sensitivity and specificity of GRA1-based on ELISA were 100% and 86.36%, positive prediction value (ppv was 94.11%. These data indicate that the recombinant protein GRA1 is a highly immunogenic protein in human toxoplasmosis and become a promising marker for the screening of toxoplasmosis.

Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

International Nuclear Information System (INIS)

Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

1986-01-01

The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes 14 C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg 2+ . Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-γ-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated

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)

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

Science.gov (United States)

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

Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

International Nuclear Information System (INIS)

Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

1986-01-01

The H + translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ ≅ 70,000 and ≅ 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[ 14 C]ethylmaleimide and 7-chloro-4-nitro[ 14 C]benzo-2-oxa-1,3-diazole, labeled the M/sub r/ ≅ 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[ 14 C]dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ ≅ 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10 5 , 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ ≅ 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F 0 F 1 ATPases

Yeast three-hybrid screen identifies TgBRADIN/GRA24 as a negative regulator of Toxoplasma gondii bradyzoite differentiation.

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Anahi V Odell

Full Text Available Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

The membrane-associated form of α(s1-casein interacts with cholesterol-rich detergent-resistant microdomains.

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Annabelle Le Parc

Full Text Available Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1-casein. These experiments reveal that the insolubility of α(s1-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.

The Membrane-Associated Form of αs1-Casein Interacts with Cholesterol-Rich Detergent-Resistant Microdomains

Science.gov (United States)

Le Parc, Annabelle; Honvo Houéto, Edith; Pigat, Natascha; Chat, Sophie; Leonil, Joëlle; Chanat, Eric

2014-01-01

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that αs1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of αs1-casein in rat mammary epithelial cells. Using metabolic labelling we show that αs1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of αs1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of αs1-casein. These experiments reveal that the insolubility of αs1-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of αs1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells. PMID:25549363

The membrane-associated form of α(s1)-casein interacts with cholesterol-rich detergent-resistant microdomains.

Science.gov (United States)

Le Parc, Annabelle; Honvo Houéto, Edith; Pigat, Natascha; Chat, Sophie; Leonil, Joëlle; Chanat, Eric

2014-01-01

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1)-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1)-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1)-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1)-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1)-casein. These experiments reveal that the insolubility of α(s1)-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1)-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.

Establishing a Global Radiation Oncology Collaboration in Education (GRaCE)

DEFF Research Database (Denmark)

Turner, Sandra; Eriksen, Jesper G; Trotter, Theresa

2015-01-01

Representatives from countries and regions world-wide who have implemented modern competency-based radiation- or clinical oncology curricula for training medical specialists, met to determine the feasibility and value of an ongoing international collaboration. In this forum, educational leaders...... with similar goals, would provide a valuable vehicle to ensure training program currency, through sharing of resources and expertise, and enhance high quality radiation oncology education. Potential projects for the Global Radiation Oncology Collaboration in Education (GRaCE) were agreed upon...

Application of GRA for Sustainable Material Selection and Evaluation Using LCA

Science.gov (United States)

Jayakrishna, Kandasamy; Vinodh, Sekar; Sakthi Sanghvi, Vijayaselvan; Deepika, Chinadurai

2016-07-01

Material selection is identified as a successful key parameter in establishing any product to be sustainable, considering its end of life (EoL) characteristics. An accurate understanding of expected service conditions and environmental considerations are crucial in the selection of material plays a vital role with overwhelming customer expectations and stringent laws. Therefore, this article presents an integrated approach for sustainable material selection using grey relational analysis (GRA) considering the EoL disposal strategies with respect to an automotive product. GRA, an impact evaluation model measures the degree of similarity between the comparability (choice of material) sequence and reference (EoL strategies) sequence based on the relational grade. The ranking result shows that the outranking relationships in the order, ABS-REC > PP-INC > AL-REM > PP-LND > ABS-LND > ABS-INC > PU-LND > AL-REC > AL-LND > PU-INC > AL-INC. The best sustainable material selected was ABS and recycling was selected as the best EoL strategy with the grey relational value of 2.43856. The best material selected by this approach, ABS was evaluated for its viability using life cycle assessment and the estimated impacts also proved the practicability of the selected material highlighting the focus on dehumidification step in the manufacturing of the case product using this developed multi-criteria approach.

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.

Construction Projects Assessment Based on the Sustainable Development Criteria by an Integrated Fuzzy AHP and Improved GRA Model

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Seyed Morteza Hatefi

2018-03-01

Full Text Available Due to the increasing population and earth pollution, managing construction and infrastructure projects with less damage to the environment and less pollution is very important. Sustainable development aims at reducing damage to the environment, making projects economical, and increasing comfort and social justice. This study proposes fuzzy analytic hierarchy process (AHP and improved grey relational analysis (GRA to assess construction projects based on the sustainable development criteria. For doing so, sustainable development criteria are first identified in economic, social, and environmental dimensions using literature review, and are then customized for urban construction projects using experts’ opinions. After designing questionnaires and collecting data, fuzzy AHP is used for determining the importance of sustainable development criteria and their subcriteria. Then, improved GRA is employed for assessing six recreational, commercial, and official centers in Isfahan regarding the weights of criteria and subcriteria. The proposed fuzzy AHP-improved GRA help us to prioritize construction projects based on the sustainable development criteria. The results of applying fuzzy AHP show that the weights of economic, social, and environmental criteria are equal to 0.330, 0.321, and 0.349, respectively, which are close to each other. This means that the importance of all three aspects of sustainability is almost equal to each other. Furthermore, “Having profits for the society”, “Increasing social justice”, and “Adherence to environmental policies” are identified as the most important indicators of sustainable development in terms of economic, social, and environmental aspects, respectively. Finally, the results of employing improved GRA determine Negin Chaharbagh recreational and commercial complex as the best project.

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...

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

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....

Identification of membrane-associated proteins with pathogenic potential expressed by Corynebacterium pseudotuberculosis grown in animal serum.

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Raynal, José Tadeu; Bastos, Bruno Lopes; Vilas-Boas, Priscilla Carolinne Bagano; Sousa, Thiago de Jesus; Costa-Silva, Marcos; de Sá, Maria da Conceição Aquino; Portela, Ricardo Wagner; Moura-Costa, Lília Ferreira; Azevedo, Vasco; Meyer, Roberto

2018-01-25

Previous works defining antigens that might be used as vaccine targets against Corynebacterium pseudotuberculosis, which is the causative agent of sheep and goat caseous lymphadenitis, have focused on secreted proteins produced in a chemically defined culture media. Considering that such antigens might not reflect the repertoire of proteins expressed during infection conditions, this experiment aimed to investigate the membrane-associated proteins with pathogenic potential expressed by C. pseudotuberculosis grown directly in animal serum. Its membrane-associated proteins have been extracted using an organic solvent enrichment methodology, followed by LC-MS/MS and bioinformatics analysis for protein identification and classification. The results revealed 22 membrane-associated proteins characterized as potentially pathogenic. An interaction network analysis indicated that the four potentially pathogenic proteins ciuA, fagA, OppA4 and OppCD were biologically connected within two distinct network pathways, which were both associated with the ABC Transporters KEGG pathway. These results suggest that C. pseudotuberculosis pathogenesis might be associated with the transport and uptake of nutrients; other seven identified potentially pathogenic membrane proteins also suggest that pathogenesis might involve events of bacterial resistance and adhesion. The proteins herein reported potentially reflect part of the protein repertoire expressed during real infection conditions and might be tested as vaccine antigens.

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 ...

Optimization of Maghemite (γ-Fe2O3) Nano-Powder Mixed micro-EDM of CoCrMo with Multiple Responses Using Gray Relational Analysis (GRA)

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Mejid Elsiti, Nagwa; Noordin, M. Y.; Idris, Ani; Saed Majeed, Faraj

2017-10-01

This paper presents an optimization of process parameters of Micro-Electrical Discharge Machining (EDM) process with (γ-Fe2O3) nano-powder mixed dielectric using multi-response optimization Grey Relational Analysis (GRA) method instead of single response optimization. These parameters were optimized based on 2-Level factorial design combined with Grey Relational Analysis. The machining parameters such as peak current, gap voltage, and pulse on time were chosen for experimentation. The performance characteristics chosen for this study are material removal rate (MRR), tool wear rate (TWR), Taper and Overcut. Experiments were conducted using electrolyte copper as the tool and CoCrMo as the workpiece. Experimental results have been improved through this approach.

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.

Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters.

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Sloothaak, J; Odoni, D I; de Graaff, L H; Martins Dos Santos, V A P; Schaap, P J; Tamayo-Ramos, J A

2015-01-01

The development of biological processes that replace the existing petrochemical-based industry is one of the biggest challenges in biotechnology. Aspergillus niger is one of the main industrial producers of lignocellulolytic enzymes, which are used in the conversion of lignocellulosic feedstocks into fermentable sugars. Both the hydrolytic enzymes responsible for lignocellulose depolymerisation and the molecular mechanisms controlling their expression have been well described, but little is known about the transport systems for sugar uptake in A. niger. Understanding the transportome of A. niger is essential to achieve further improvements at strain and process design level. Therefore, this study aims to identify and classify A. niger sugar transporters, using newly developed tools for in silico and in vivo analysis of its membrane-associated proteome. In the present research work, a hidden Markov model (HMM), that shows a good performance in the identification and segmentation of functionally validated glucose transporters, was constructed. The model (HMMgluT) was used to analyse the A. niger membrane-associated proteome response to high and low glucose concentrations at a low pH. By combining the abundance patterns of the proteins found in the A. niger plasmalemma proteome with their HMMgluT scores, two new putative high-affinity glucose transporters, denoted MstG and MstH, were identified. MstG and MstH were functionally validated and biochemically characterised by heterologous expression in a S. cerevisiae glucose transport null mutant. They were shown to be a high-affinity glucose transporter (K m = 0.5 ± 0.04 mM) and a very high-affinity glucose transporter (K m = 0.06 ± 0.005 mM), respectively. This study, focusing for the first time on the membrane-associated proteome of the industrially relevant organism A. niger, shows the global response of the transportome to the availability of different glucose concentrations. Analysis of the A. niger

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 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.

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

Science.gov (United States)

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.

Avaliação de processos de misturas de concretos com agregados graúdos reciclados

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Luciana de Nazaré Pinheiro Cordeiro

Full Text Available Resumo A reutilização dos resíduos de construção civil vem sendo uma estratégia do setor para alcançar a sustentabilidade, mesmo sendo aplicada em um pequeno volume de obras. A variabilidade do resíduo de concreto é uma condição inerente do produto, que precisa ser levada em consideração, e o controle do processo produtivo de concretos produzidos com resíduos de concreto pode ser uma maneira de majorar a qualidade do compósito final. Nesse sentido, este artigo tem por objetivo avaliar a eficiência do processo de mistura e do tipo de misturador ante a fragilidade do desempenho de concretos com agregados graúdos reciclados de concreto e verificar a influência na resistência à compressão. Como objetivo específico tem-se o efeito do misturador no desempenho do concreto. O agregado graúdo reciclado de concreto foi usado em substituição total ao agregado graúdo natural. E os resultados mostraram que não há diferença significativa entre os concretos produzidos a partir de diferentes ordens de misturas; ambas promoveram resultados satisfatórios de resistência à compressão na idade de 28 dias. Porém, as misturas realizadas em misturador de eixo vertical se mostraram mais eficientes que as misturas produzidas em betoneiras de eixo inclinado, uma vez que os concretos produzidos pelas primeiras se mostram com menor coeficiente de variação. Acredita-se que as características do equipamento contribuíram para uma melhor homogeneidade, levando à menor variabilidade nos resultados do concreto.

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...

Nutrient-dependent methylation of a membrane-associated protein of Escherichia coli

International Nuclear Information System (INIS)

Young, C.C.; Alvarez, J.D.; Bernlohr, R.W.

1990-01-01

Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and [methyl-3H]methionine. The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process. Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43. P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues. The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B. licheniformis and are consistent with the proposal that methylation of this protein functions in nutrient sensing

Physiological implications of seasonal variation in membrane-associated calcium in red spruce mesophyll cells

Science.gov (United States)

D.H. DeHayes; P.G. Schaberg; G.J. Hawley; C.H. Borer; J.R. Cumming; J.R. Strimbeck

1997-01-01

We examined the pattern of seasonal variation in total foliar calcium (Ca) pools and plasma membrane-associated Ca (mCa) in mesophyll cells of current-year and 1-year-old needles of red spruce (Picea rubens Sarg.) and the relationship between mCa and total foliar Ca on an individual plant and seasonal basis. Foliar samples were collected from...

Diagnostic value of a Rec-ELISA using Toxoplasma gondii recombinant SporoSAG, BAG1, and GRA1 proteins in murine models infected orally with tissue cysts and oocysts.

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Mert Döşkaya

Full Text Available Toxoplasma gondii causes congenital toxoplasmosis in newborns resulting with fetal anomalies. Determining the initiation time of infection is very important for pregnant women and current serological assays have drawbacks in distinguishing the recently acute toxoplasmosis. Diagnosis of recently acute infection may be improved by using stage specific antigens in serological assays. In the present study, the diagnostic value of sporozoite specific SporoSAG, bradyzoite specific BAG1 proteins and GRA1 protein expressed by all forms of the parasite have been evaluated ELISA using sera systematically collected from mice administered orally with tissue cyst and oocysts. The anti-SporoSAG IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 1, 10, and 15 (P<0.01. The anti-BAG1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly at days 15, 40, and 120 (P<0.05. The anti-GRA1 IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 2, 10, and 40 (P<0.01. The anti-GRA1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly only at day 40 (P<0.05. The anti-SporoSAG, anti-BAG1, and anti-GRA1 IgG titers of mice showed significant increases at day 40 (P<0.05 and decrement started for only anti-GRA1 IgG at day 120. The presence of anti-SporoSAG IgM and IgG antibodies can be interpreted as recently acute infection between days 10-40 because IgM decreases at day 40. Similarly, presence of anti-BAG1 IgM and absence of IgG can be evaluated as a recently acute infection that occurred 40 days before because IgG peaks at day 40. A peak in anti-GRA1 antibody level at first testing and reduction in consecutive sample can be considered as an infection approximately around day 40 or prior. Overall, recombinant SporoSAG, BAG1 and GRA1 proteins can be accepted as valuable diagnostic markers of recently acute toxoplasmosis.

Toxoplasma gondii-derived synthetic peptides containing B- and T-cell epitopes from GRA2 protein are able to enhance mice survival in a model of experimental toxoplasmosis

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Luciana Machado Bastos

2016-06-01

Full Text Available Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2 is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN, as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b, mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-alpha and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

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.

Rapid time-resolved magnetic resonance angiography via a multiecho radial trajectory and GraDeS reconstruction.

Science.gov (United States)

Lee, Gregory R; Seiberlich, Nicole; Sunshine, Jeffrey L; Carroll, Timothy J; Griswold, Mark A

2013-02-01

Contrast-enhanced magnetic resonance angiography is challenging due to the need for both high spatial and temporal resolution. A multishot trajectory composed of pseudo-random rotations of a single multiecho radial readout was developed. The trajectory is designed to give incoherent aliasing artifacts and a relatively uniform distribution of projections over all time scales. A field map (computed from the same data set) is used to avoid signal dropout in regions of substantial field inhomogeneity. A compressed sensing reconstruction using the GraDeS algorithm was used. Whole brain angiograms were reconstructed at 1-mm isotropic resolution and a 1.1-s frame rate (corresponding to an acceleration factor > 100). The only parameter which must be chosen is the number of iterations of the GraDeS algorithm. A larger number of iterations improves the temporal behavior at cost of decreased image signal-to-noise ratio. The resulting images provide a good depiction of the cerebral vasculature and have excellent arterial/venous separation. Copyright © 2012 Wiley Periodicals, Inc.

Human skin basement membrane-associated heparan sulphate proteoglycan: distinctive differences in ultrastructural localization as a function of developmental age

DEFF Research Database (Denmark)

Horiguchi, Y; Fine, J D; Couchman, J R

1991-01-01

was identical to that observed in neonatal and adult human skin. These findings demonstrate that active remodelling of the dermo-epidermal junction occurs during at least the first two trimesters, and affects not only basement membrane-associated structures but also specific antigens.......Recent studies have demonstrated that skin basement membrane components are expressed within the dermo-epidermal junction in an orderly sequence during human foetal development. We have investigated the ultrastructural localization of basement membrane-related antigens in human foetal skin...... at different developmental ages using two monoclonal antibodies to a well-characterized basement membrane-associated heparan sulphate proteoglycan. A series of foetal skin specimens (range, 54-142 gestational days) were examined using an immunoperoxidase immunoelectron microscopic technique. In specimens...

Prioritizing the performance of civil development projects in governmental administration agencies, using gray relational analysis (GRA and TOPSIS approach

Directory of Open Access Journals (Sweden)

Ali Mohammadi

2016-06-01

Full Text Available A key indicator to evaluate the success of an organization is the degree of meeting specific civil project goals based on a predetermined schedule. Therefore, the main purpose of this paper is to evaluate the performance of governmental administration agencies based on realization of civil project goals. In this paper, the information published by the President Deputy of Strategic Planning and Control, that publishes an annual report of evaluation indicators for national civil development projects, are used to evaluate and prioritize the major and non-major governmental agencies. Also, the Gray Relational Analysis (GRA and the TOPSIS method are employed to analyze the data. The results indicate that using the GRA method, Supreme Council of Seminary and using the TOPSIS method, The Ministry of Labor and Social Affaires have gained the highest ranking.

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

Energy Technology Data Exchange (ETDEWEB)

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

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

Science.gov (United States)

Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

2005-07-01

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

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

Science.gov (United States)

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.

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.

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

Science.gov (United States)

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

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 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

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.

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...

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.

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

Science.gov (United States)

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.

O teatro da Graça na segunda metade do século XVIII

OpenAIRE

Gomes, Francisco Luís Rosa Ferreira

2012-01-01

Tese de mestrado, Estudos de Teatro, Universidade de Lisboa, Faculdade de Letras, 2012 Teatro de história breve, o Teatro da Graça, sobre o qual escasseia informação, é o objecto de estudo deste trabalho, que visa reconstruir, na medida do possível, aspectos relacionados com a sua existência no decurso da segunda metade do século XVIII. Procurámos, ao longo do trabalho realizado, clarificar detalhes e pormenores relativos a esta casa de teatro. A saber, características relativa...

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.

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.

Hermann Günther Graßmann (1809–1877) Visionary Mathematician, Scientist and Neohumanist Scholar : Papers from a Sesquicentennial Conference

CERN Document Server

1996-01-01

In this volume specialists in mathematics, physics, and linguistics present the first comprehensive analysis of the ideas and influence of Hermann G. Graßmann (1809-1877), the remarkable universalist whose work recast the foundations of these disciplines and shaped the course of their modern development.

Phosphoenolpyruvate-Dependent Fructose Phosphotransferase System of Rhodopseudomonas sphaeroides : Purification and Physicochemical and Immunochemical Characterization of a Membrane-Associated Enzyme I

NARCIS (Netherlands)

Brouwer, Marius; Elferink, Marieke G.L.; Robillard, George T.

1982-01-01

The phosphotransferase system (PTS) of the phototrophic bacterium Rhodopseudomonas sphaeroides consists of a component located in the cytoplasmic membrane and a membrane-associated enzyme called “soluble factor” (SF). SF has been partially purified by a combination of hydrophobic interaction and

Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

International Nuclear Information System (INIS)

Semelr, B.L.; Anderson, C.W.; Hanecak, R.; Dorner, L.F.; Wimmer, E.

1982-01-01

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNA sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex

Expression of membrane-associated proteins within single emulsion cell facsimiles.

Science.gov (United States)

Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

2012-07-07

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

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

[Function of transport H+-ATPases in plant cell plasma and vacuolar membranes of maize under salt stress conditions and effect of adaptogenic preparations].

Science.gov (United States)

Rybchenko, Zh I; Palladina, T O

2011-01-01

Participations of electrogenic H+-pumps of plasma and vacuolar membranes represented by E1-E2 and V-type H+-ATPases in plant cell adaptation to salt stress conditions has been studied by determination of their transport activities. Experiments were carried out on corn seedlings exposed during 1 or 10 days at 0.1 M NaCl. Preparations Methyure and Ivine were used by seed soaking at 10(-7) M. Plasma and vacuolar membrane fractions were isolated from corn seedling roots. In variants without NaCl a hydrolytical activity of plasma membrane H+-ATPase was increased with seedling age and its transport one was changed insignificantly, wherease the response of the weaker vacuolar H+-ATPase was opposite. NaCl exposition decreased hydrolytical activities of both H+-ATPases and increased their transport ones. These results demonstrated amplification of H+-pumps function especially represented by vacuolar H+-ATPase. Both preparations, Methyure mainly, caused a further increase of transport activity which was more expressed in NaCl variants. Obtained results showed the important role of these H+-pumps in plant adaptation under salt stress conditions realized by energetical maintenance of the secondary active Na+/H+ -antiporters which remove Na+ from cytoplasm.

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

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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.

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...

Optimum design of brake friction material using hybrid entropy-GRA approach

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Kumar Naresh

2016-01-01

Full Text Available The effect of Kevlar and natural fibres on the performance of brake friction materials was evaluated. Four friction material specimens were developed by varying the proportion of Kevlar and natural fibres. Two developed composite contained 5-10 wt.% of Kevlar fibre while in the other two the Kevlar fibre was replaced with same amount of natural fibre. SAE J661 protocol was used for the assessment of the tribological properties on a Chase testing machine. Result shows that the specimens containing Kevlar fibres shows higher friction and wear performance, whereas Kevlar replacement with natural fibre resulted in improved fade, recovery and friction fluctuations. Further hybrid entropy-GRA (grey relation analysis approach was applied to select the optimal friction materials using various performance defining attributes (PDA including friction, wear, fade, recovery, friction fluctuations and cost. The friction materials with 10 wt% of natural fibre exhibited the best overall quality.

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

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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.

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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

Role of plasma membrane-associated AKAPs for the regulation of cardiac IK1 current by protein kinase A.

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Seyler, Claudia; Scherer, Daniel; Köpple, Christoph; Kulzer, Martin; Korkmaz, Sevil; Xynogalos, Panagiotis; Thomas, Dierk; Kaya, Ziya; Scholz, Eberhard; Backs, Johannes; Karle, Christoph; Katus, Hugo A; Zitron, Edgar

2017-05-01

The cardiac I K1 current stabilizes the resting membrane potential of cardiomyocytes. Protein kinase A (PKA) induces an inhibition of I K1 current which strongly promotes focal arrhythmogenesis. The molecular mechanisms underlying this regulation have only partially been elucidated yet. Furthermore, the role of A-kinase anchoring proteins (AKAPs) in this regulation has not been examined to date. The objective of this project was to elucidate the molecular mechanisms underlying the inhibition of I K1 by PKA and to identify novel molecular targets for antiarrhythmic therapy downstream β-adrenoreceptors. Patch clamp and voltage clamp experiments were used to record currents and co-immunoprecipitation, and co-localization experiments were performed to show spatial and functional coupling. Activation of PKA inhibited I K1 current in rat cardiomyocytes. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide inhibitor AKAP-IS. We observed functional and spatial coupling of the plasma membrane-associated AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channel subunits, but not to Kir2.3 channels. In contrast, AKAPyotiao had no functional effect on the PKA regulation of Kir channels. AKAP15 and AKAP79 co-immunoprecipitated with and co-localized to Kir2.1 and Kir2.2 channel subunits in ventricular cardiomyocytes. In this study, we provide evidence for coupling of cardiac Kir2.1 and Kir2.2 subunits with the plasma membrane-bound AKAPs 15 and 79. Cardiac membrane-associated AKAPs are a functionally essential part of the regulatory cascade determining I K1 current function and may be novel molecular targets for antiarrhythmic therapy downstream from β-adrenoreceptors.

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.

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

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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

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

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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 remodeling and an altered membrane-associated proteome may drive the differential effects of EPA and DHA treatment on skeletal muscle glucose uptake and protein accretion.

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Jeromson, Stewart; Mackenzie, Ivor; Doherty, Mary K; Whitfield, Phillip D; Bell, Gordon; Dick, James; Shaw, Andy; Rao, Francesco V; Ashcroft, Stephen P; Philp, Andrew; Galloway, Stuart D R; Gallagher, Iain; Hamilton, D Lee

2018-06-01

In striated muscle, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C 2 C 12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment of the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. Fatty acid methyl esters (FAME) analysis revealed that both EPA and DHA led to similar but substantials changes in fatty acid profiles with the exception of arachidonic acid, which was decreased only by DHA, and docosapentanoic acid (DPA), which was increased only by EPA treatment. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long-chain fatty acids with five (EPA treatment) or six (DHA treatment) double bonds. As these are typically membrane-associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane-associated proteome. We conclude that the EPA-specific increase in polyunsaturated long-chain fatty acids in the phospholipid fraction is associated with an altered membrane-associated proteome and these may be critical events in the metabolic remodeling induced by EPA treatment.

The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

Science.gov (United States)

Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

2016-01-01

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

Impact of Kishnica and Badovci Flotation Tailing Dams on Levels of Heavy Metals in Water of Graçanica River (Kosovo

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Fatbardh Gashi

2017-01-01

Full Text Available The main objective of this study was to perform assessment of water quality of Graçanica River (Kosovo, impacted by Kishnica and Badovci flotation tailing dams, using ICP-OES method. The obtained results show that the mean values of all heavy metals in studied river water samples were significantly high, with following maximal concentrations: As (0.033 mgL−1, Cd (0.002 mgL−1, Cr (0.225 mgL−1, Cu (0.015 mgL−1, Hg (0.004 mgL−1, Mn (15.66 mgL−1, Ni (0.255 mgL−1, Pb (0.013 mgL−1, and Zn (0.612 mgL−1, but only two samples from locations influenced by Kishnica and Badovci flotation tailing dams showed statistically anomalous values of Cr3+, Cu2+, Mn2+, Zn2+, and Hg2+. According to assessment based on Croatian standards, locations near both flotation tailing dams are significantly polluted with majority of studied metals, while downstream sampling stations are almost unpolluted or slightly polluted. Mercury is found to be the most significant contaminant. According to WHO recommended values for drinking water, on all locations values were within the limits for Al, Cd, Cu, and Zn, while for As, Cr, Hg, Mn, Ni, and Pb values exceed recommended values on some sampling stations. Further monitoring of water and possibly sediments of Graçanica River is advised, as well as performing of remediation of Kishnica and Badovci mine tailing dams.

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.

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.

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.

Science.gov (United States)

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.

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

Science.gov (United States)

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

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

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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.

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

Science.gov (United States)

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

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

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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

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.

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

Directory of Open Access Journals (Sweden)

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.

Revealing mechanisms of selective, concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase.

Science.gov (United States)

Bauer, Georg; Zarkovic, Neven

2015-04-01

Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H2O2 and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

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.

Integrated Supplier Selection Framework in a Resilient Construction Supply Chain: An Approach via Analytic Hierarchy Process (AHP and Grey Relational Analysis (GRA

Directory of Open Access Journals (Sweden)

Ting-Kwei Wang

2017-02-01

Full Text Available Construction supply chain management is a unique and problematic issue within the construction industry due to its inevitable external risks and variations. The resilience capability of a supplier is of significance in supplier selection; a supplier selected in the context of a resilient construction supply chain (RCSC is referred to in this research as a “resilient construction supplier”. This paper proposes a supplier selection framework tailored to effective information integration for supply chain management. The proposed framework works by integrating building information modeling (BIM and a geographic information system (GIS in a RCSC. BIM and GIS together provide highly transparent construction material information, enhanced supply chain status visualization, and workable access information for supplier selection. Supplier performance is evaluated via seventeen resilient criteria under a combined methodology consisting of the analytic hierarchy process (AHP and grey relational analysis (GRA; AHP and GRA weigh the criteria and rank the suppliers respectively. By varying the weightings given to each criterion, sensitivity analysis was conducted to identify the criteria of resilience which impact the selection priorities of suppliers. An illustrative example is also provided to show the overall process of the proposed framework.

GraDit: graph-based data repair algorithm for multiple data edits rule violations

Science.gov (United States)

Ode Zuhayeni Madjida, Wa; Gusti Bagus Baskara Nugraha, I.

2018-03-01

Constraint-based data cleaning captures data violation to a set of rule called data quality rules. The rules consist of integrity constraint and data edits. Structurally, they are similar, where the rule contain left hand side and right hand side. Previous research proposed a data repair algorithm for integrity constraint violation. The algorithm uses undirected hypergraph as rule violation representation. Nevertheless, this algorithm can not be applied for data edits because of different rule characteristics. This study proposed GraDit, a repair algorithm for data edits rule. First, we use bipartite-directed hypergraph as model representation of overall defined rules. These representation is used for getting interaction between violation rules and clean rules. On the other hand, we proposed undirected graph as violation representation. Our experimental study showed that algorithm with undirected graph as violation representation model gave better data quality than algorithm with undirected hypergraph as representation model.

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.

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...

A comparison of the imaging characteristics of the new Kodak Hyper Speed G film with the current T-MAT G/RA film and the CR 9000 system.

Science.gov (United States)

Monnin, P; Gutierrez, D; Bulling, S; Lepori, D; Verdun, F R

2005-10-07

Three standard radiation qualities (RQA 3, RQA 5 and RQA 9) and two screens, Kodak Lanex Regular and Insight Skeletal, were used to compare the imaging performance and dose requirements of the new Kodak Hyper Speed G and the current Kodak T-MAT G/RA medical x-ray films. The noise equivalent quanta (NEQ) and detective quantum efficiencies (DQE) of the four screen-film combinations were measured at three gross optical densities and compared with the characteristics for the Kodak CR 9000 system with GP (general purpose) and HR (high resolution) phosphor plates. The new Hyper Speed G film has double the intrinsic sensitivity of the T-MAT G/RA film and a higher contrast in the high optical density range for comparable exposure latitude. By providing both high sensitivity and high spatial resolution, the new film significantly improves the compromise between dose and image quality. As expected, the new film has a higher noise level and a lower signal-to-noise ratio than the standard film, although in the high frequency range this is compensated for by a better resolution, giving better DQE results--especially at high optical density. Both screen-film systems outperform the phosphor plates in terms of MTF and DQE for standard imaging conditions (Regular screen at RQA 5 and RQA 9 beam qualities). At low energy (RQA 3), the CR system has a comparable low-frequency DQE to screen-film systems when used with a fine screen at low and middle optical densities, and a superior low-frequency DQE at high optical density.

A comparison of the imaging characteristics of the new Kodak Hyper Speed G film with the current T-MAT G/RA film and the CR 9000 system

Energy Technology Data Exchange (ETDEWEB)

Monnin, P [Institut universitaire de Radiophysique Appliquee (IRA), CH-1007 Lausanne (Switzerland); Gutierrez, D [Institut universitaire de Radiophysique Appliquee (IRA), CH-1007 Lausanne (Switzerland); Bulling, S [Institut universitaire de Radiophysique Appliquee (IRA), CH-1007 Lausanne (Switzerland); Lepori, D [Department of Radiology, University Hospital Center (CHUV), CH-1011 Lausanne (Switzerland); Verdun, F R [Institut universitaire de Radiophysique Appliquee (IRA), CH-1007 Lausanne (Switzerland)

2005-10-07

Three standard radiation qualities (RQA 3, RQA 5 and RQA 9) and two screens, Kodak Lanex Regular and Insight Skeletal, were used to compare the imaging performance and dose requirements of the new Kodak Hyper Speed G and the current Kodak T-MAT G/RA medical x-ray films. The noise equivalent quanta (NEQ) and detective quantum efficiencies (DQE) of the four screen-film combinations were measured at three gross optical densities and compared with the characteristics for the Kodak CR 9000 system with GP (general purpose) and HR (high resolution) phosphor plates. The new Hyper Speed G film has double the intrinsic sensitivity of the T-MAT G/RA film and a higher contrast in the high optical density range for comparable exposure latitude. By providing both high sensitivity and high spatial resolution, the new film significantly improves the compromise between dose and image quality. As expected, the new film has a higher noise level and a lower signal-to-noise ratio than the standard film, although in the high frequency range this is compensated for by a better resolution, giving better DQE results-especially at high optical density. Both screen-film systems outperform the phosphor plates in terms of MTF and DQE for standard imaging conditions (Regular screen at RQA 5 and RQA 9 beam qualities). At low energy (RQA 3), the CR system has a comparable low-frequency DQE to screen-film systems when used with a fine screen at low and middle optical densities, and a superior low-frequency DQE at high optical density.

A Combined Fuzzy-AHP and Fuzzy-GRA Methodology for Hydrogen Energy Storage Method Selection in Turkey

Directory of Open Access Journals (Sweden)

Aytac Yildiz

2013-06-01

Full Text Available In this paper, we aim to select the most appropriate Hydrogen Energy Storage (HES method for Turkey from among the alternatives of tank, metal hydride and chemical storage, which are determined based on expert opinions and literature review. Thus, we propose a Buckley extension based fuzzy Analytical Hierarchical Process (Fuzzy-AHP and linear normalization based fuzzy Grey Relational Analysis (Fuzzy-GRA combined Multi Criteria Decision Making (MCDM methodology. This combined approach can be applied to a complex decision process, which often makes sense with subjective data or vague information; and used to solve to solve HES selection problem with different defuzzification methods. The proposed approach is unique both in the HES literature and the MCDM literature.

À "frente" da Semana de Arte Moderna: a presença de Graça Aranha e Paulo Prado

OpenAIRE

Waldman,Thaís

2010-01-01

Reconhecidos como personagens centrais pelo grupo de intelectuais e artistas ligados à Semana de Arte Moderna de 1922, Graça Aranha e Paulo Prado são muitas vezes deixados de lado pelos estudiosos e, em geral, se fazem presentes nas análises de bastidores e/ou em referências de terceiros. Este artigo pretende observar mais atentamente suas redes de sociabilidade e inserção, o que revela, entre outras coisas, as ambivalências do engate de ambos os autores em um projeto "moderno", assim como ce...

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.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

Science.gov (United States)

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

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.

Renal Tumor Cryoablation Planning. The Efficiency of Simulation on Reconstructed 3D CT Scan

Directory of Open Access Journals (Sweden)

Ciprian Valerian LUCAN

2010-12-01

Full Text Available Introduction & Objective: Nephron-sparing surgical techniques risks are related to tumor relationships with adjacent anatomic structures. Complexity of the renal anatomy drives the interest to develop tools for 3D reconstruction and surgery simulation. The aim of the article was to assess the simulation on reconstructed 3D CT scan used for planning the cryoablation. Material & Method: A prospective randomized study was performed between Jan. 2007 and July 2009 on 27 patients who underwent retroperitoneoscopic T1a renal tumors cryoablation (RC. All patients were assessed preoperatively by CT scan, also used for 3D volume rendering. In the Gr.A, the patients underwent surgery planning by simulation on 3D CT scan. In the Gr.B., patients underwent standard RC. The two groups were compared in terms of surgical time, bleeding, postoperative drainage, analgesics requirement, hospital stay, time to socio-professional reintegration. Results: Fourteen patients underwent preoperative cryoablation planning (Gr.A and 13 patients underwent standard CR (Gr.B. All parameters analyzed were shorter in the Gr.A. On multivariate logistic regression, only shortens of the surgical time (138.79±5.51 min. in Gr.A. vs. 140.92±5.54 min in Gr.B. and bleeding (164.29±60.22 mL in Gr.A. vs. 215.38±100.80 mL in Gr.B. achieved statistical significance (p<0.05. The number of cryoneedles assessed by simulation had a 92.52% accuracy when compared with those effectively used. Conclusions: Simulation of the cryoablation using reconstructed 3D CT scan improves the surgical results. The application used for simulation was able to accurately assess the number of cryoneedles required for tumor ablation, their direction and approach.

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

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

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.

The plasma membrane-associated NADH oxidase of spinach leaves responds to blue light

Science.gov (United States)

Morre, D. James; Penel, Claude; Greppin, Hubert; Morre, Dorothy M.

2002-01-01

The plasma membrane-associated NADH oxidase (NOX) of spinach leaf disks is characterized by oscillations in activity with a regular period length of ca. 24 min. Within a single population of plants exposed to light at the same time, NOX activities of all plants function synchronously. Exposure of plants transferred from darkness to blue light (495 nm, 2 min, 50 micromoles m-2 s-1) resulted in a complex response pattern but with a new maximum in the rate of NOX activity 36 (24+12) min after illumination and then with maxima in the rate of NOX activity every 24 min thereafter. Transient maxima in NOX activity were observed as well after 9.3 + /- 1.4 and 20.7 +/- 2.1 min. The blue light response differed from the response to red (650 nm, 10 min, 50 micromoles m-2 s-1) or white light where activity maxima were initiated 12 min after the light exposure followed by maxima every 24 min thereafter. Green or yellow light was ineffective. The light response was independent of the time in the 24-min NOX cycle when the light was given. The net effects of blue and red light were ultimately the same with a new maximum in the rate of NOX activity at 12+24=36 min (and every 24 min thereafter), but the mechanisms appear to be distinct.

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

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

Science.gov (United States)

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

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

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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...

Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

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The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

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

Science.gov (United States)

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.

Integrating solid-state NMR and computational modeling to investigate the structure and dynamics of membrane-associated ghrelin.

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Gerrit Vortmeier

Full Text Available The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide's secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide's positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8-17 form an α-helix, while residues 21-23 and 26-27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane.

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

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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...

À "frente" da Semana de Arte Moderna: a presença de Graça Aranha e Paulo Prado

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Thaís Waldman

2010-06-01

Full Text Available Reconhecidos como personagens centrais pelo grupo de intelectuais e artistas ligados à Semana de Arte Moderna de 1922, Graça Aranha e Paulo Prado são muitas vezes deixados de lado pelos estudiosos e, em geral, se fazem presentes nas análises de bastidores e/ou em referências de terceiros. Este artigo pretende observar mais atentamente suas redes de sociabilidade e inserção, o que revela, entre outras coisas, as ambivalências do engate de ambos os autores em um projeto "moderno", assim como certas ambivalências presentes no interior do próprio movimento modernista.

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.

Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

Energy Technology Data Exchange (ETDEWEB)

Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

2011-12-16

Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

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

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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.

Membrane-associated IL 1-like activity on rat dendritic cells

International Nuclear Information System (INIS)

Nagelkerken, L.M.; van Breda Vriesman, P.J.C.

1986-01-01

The secretion of interleukin 1 (IL 1) by rat dendritic cells (DC) was studied in relation to their ability to induce the production interleukin 2 (IL 2 ) and to induce IL 2 responsiveness. IL 1 (or IL 1-like activity) was measured by its capacity to enhance IL 2 production by EL4 cells. In contrast to peritoneal exudate cells (PEC) or splenic adherent cells, DC from thoracic duct lymph (TD-DC) or from spleen did not secrete detectable amounts of IL 1 on stimulation with LPS/Silica. However, TD-DC and splenic DC were able to enhance IL 2 production by EL4 cells directly, and were only two times less effective than PEC. By preventing cell-to-cell contact between stimulator cells and EL4 cells, it was demonstrated that most of the IL 2-inducing activity of TD-DC and PEC was associated with the cell membrane. Treatment with 1% paraformaldehyde (PFA) to abolish metabolic activity resulted in a 50% decrease (or inactivation) of IL 2-inducing activity of TD-DC in the EL4 assay. Moreover, UVB-irradiation (300 mJ/cm 2 ) of TD-DC, which has been described to inhibit the release of IL 1 by macrophages, caused a 70% decrease in IL 2-inducing activity. These results suggest that membrane-associated structures, that are identical to or mimic Il 1, are involved in the activation of T cells by DC

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba δ-endotoxin complex revealed by electron crystallography: Implications for toxin-pore formation

International Nuclear Information System (INIS)

Ounjai, Puey; Unger, Vinzenz M.; Sigworth, Fred J.; Angsuthanasombat, Chanan

2007-01-01

The insecticidal nature of Cry δ-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17 A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore

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

Science.gov (United States)

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.

Integrating Solid-State NMR and Computational Modeling to Investigate the Structure and Dynamics of Membrane-Associated Ghrelin

Science.gov (United States)

Els-Heindl, Sylvia; Chollet, Constance; Scheidt, Holger A.; Beck-Sickinger, Annette G.; Meiler, Jens; Huster, Daniel

2015-01-01

The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide’s secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide’s positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8–17 form an α-helix, while residues 21–23 and 26–27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane. PMID:25803439

Application of GRA method, dynamic analysis and fuzzy set theory in evaluation and selection of emergency treatment technology for large scale phenol spill incidents

Science.gov (United States)

Zhao, Jingjing; Yu, Lean; Li, Lian

2017-05-01

Select an appropriate technology in an emergency response is a very important issue with various kinds of chemical contingency spills frequently taking place. Due to the complexity, fuzziness and uncertainties of the chemical contingency spills, the theory of GRA method, dynamic analysis combined with fuzzy set theory will be appropriately applied to selection and evaluation of emergency treatment technology. Finally, a emergency phenol spill accidence occurred in highway is provided to illustrate the applicability and feasibility of the proposed methods.

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

Science.gov (United States)

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.

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.

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.

Combination of Biorthogonal Wavelet Hybrid Kernel OCSVM with Feature Weighted Approach Based on EVA and GRA in Financial Distress Prediction

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Chao Huang

2014-01-01

Full Text Available Financial distress prediction plays an important role in the survival of companies. In this paper, a novel biorthogonal wavelet hybrid kernel function is constructed by combining linear kernel function with biorthogonal wavelet kernel function. Besides, a new feature weighted approach is presented based on economic value added (EVA and grey relational analysis (GRA. Considering the imbalance between financially distressed companies and normal ones, the feature weighted one-class support vector machine based on biorthogonal wavelet hybrid kernel (BWH-FWOCSVM is further put forward for financial distress prediction. The empirical study with real data from the listed companies on Growth Enterprise Market (GEM in China shows that the proposed approach has good performance.

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.

Establishing a Global Radiation Oncology Collaboration in Education (GRaCE): Objectives and priorities.

Science.gov (United States)

Turner, Sandra; Eriksen, Jesper G; Trotter, Theresa; Verfaillie, Christine; Benstead, Kim; Giuliani, Meredith; Poortmans, Philip; Holt, Tanya; Brennan, Sean; Pötter, Richard

2015-10-01

Representatives from countries and regions world-wide who have implemented modern competency-based radiation- or clinical oncology curricula for training medical specialists, met to determine the feasibility and value of an ongoing international collaboration. In this forum, educational leaders from the ESTRO School, encompassing many European countries adopting the ESTRO Core Curriculum, and clinician educators from Canada, Denmark, the United Kingdom, Australia and New Zealand considered the training and educational arrangements within their jurisdictions, identifying similarities and challenges between programs. Common areas of educational interest and need were defined, which included development of new competency statements and assessment tools, and the application of the latter. The group concluded that such an international cooperation, which might expand to include others with similar goals, would provide a valuable vehicle to ensure training program currency, through sharing of resources and expertise, and enhance high quality radiation oncology education. Potential projects for the Global Radiation Oncology Collaboration in Education (GRaCE) were agreed upon, as was a strategy designed to maintain momentum. This paper describes the rationale for establishing this collaboration, presents a comparative view of training in the jurisdictions represented, and reports early goals and priorities. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Failure to transmit avian vacuolar myelinopathy to mallard ducks

Science.gov (United States)

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.

Identification of the antigenic region of Neospora caninum dense granule protein 7 using ELISA.

Science.gov (United States)

Abdelbaky, Hanan H; Fereig, Ragab M; Nishikawa, Yoshifumi

2018-06-26

Dense granule protein 7 (NcGRA7) is a potent diagnostic antigen of Neospora caninum. Some studies have reported on the difficult expression, low yield, and variable degree of solubility of recombinant NcGRA7. We aimed to unravel the possible causes for these issues and tested NcGRA7 antigenicity in enzyme linked immunosorbent assays (ELISAs). The NcGRA7 coding sequence (217 amino acids) was split into five amino acid regions: NcGRA7m (27-217), NcGRA7m3 (27-160), NcGRA7m4 (27-135), NcGRA7m5 (161-190), and NcGRA7m6 (188-217). Three fragments, NcGRA7m, NcGRA7m3 and NcGRA7m4, exhibited high antigenic properties when tested against experimentally-infected mice and dog sera by ELISA. High levels of IgG2 antibodies against NcGRA7m were observed in field dog sera. In experimentally and naturally-infected cattle, the N. caninum-specific sera only reacted with NcGRA7m, indicating that this antigenic region differs among the three animal species. This study presents valuable information about the antigenic properties and topology of NcGRA7, and highlights its suitability for diagnostic purposes. Copyright © 2018. Published by Elsevier B.V.

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.

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

Science.gov (United States)

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.

Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

Directory of Open Access Journals (Sweden)

Ellen C Kammula

Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

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.

Machinability assessment of commercially pure titanium (CP-Ti) during turning operation: Application potential of GRA method

Science.gov (United States)

Khan, Akhtar; Maity, Kalipada

2018-03-01

This paper explores some of the vital machinability characteristics of commercially pure titanium (CP-Ti) grade 2. Experiments were conducted based on Taguchi’s L9 orthogonal array. The selected material was machined on a heavy duty lathe (Model: HMT NH26) using uncoated carbide inserts in dry cutting environment. The selected inserts were designated by ISO as SNMG 120408 (Model: K313) and manufactured by Kennametal. These inserts were rigidly mounted on a right handed tool holder PSBNR 2020K12. Cutting speed, feed rate and depth of cut were selected as three input variables whereas tool wear (VBc) and surface roughness (Ra) were the major attentions. In order to confirm an appreciable machinability of the work part, an optimal parametric combination was attained with the help of grey relational analysis (GRA) approach. Finally, a mathematical model was developed to exhibit the accuracy and acceptability of the proposed methodology using multiple regression equations. The results indicated that, the suggested model is capable of predicting overall grey relational grade within acceptable range.

Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

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Fei Jiang

Full Text Available Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu and heat shock protein 70 (HSP 70 are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70, IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

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

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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.

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.

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

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...

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

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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.

Decomposing series-parallel graphs into paths of length 3 and triangles

DEFF Research Database (Denmark)

Merker, Martin

2015-01-01

An old conjecture by Jünger, Reinelt and Pulleyblank states that every 2-edge-connected planar graph can be decomposed into paths of length 3 and triangles, provided its size is divisible by 3. We prove the conjecture for a class of planar graphs including all 2-edge-connected series-parallel gra...

The Antitumor Effect of Single-domain Antibodies Directed Towards Membrane-associated Catalase and Superoxide Dismutase.

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Bauer, Georg; Motz, Manfred

2016-11-01

Neutralizing single-domain antibodies directed towards catalase or superoxide dismutase (SOD) caused efficient reactivation of intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling specifically in human tumor cells. Single-domain antibodies targeted tumor cell-specific membrane-associated SOD and catalase, but not the corresponding intracellular enzymes. They were shown to be about 200-fold more effective than corresponding classical recombinant antigen-binding fragments and more than four log steps more efficient than monoclonal antibodies. Combined addition of single-domain antibodies against catalase and SOD caused a remarkable synergistic effect. Proof-of-concept experiments in immunocompromised mice using human tumor xenografts and single-domain antibodies directed towards SOD showed an inhibition of tumor growth. Neutralizing single-domain antibodies directed to catalase and SOD also caused a very strong synergistic effect with the established chemotherapeutic agent taxol, indicating an overlap of signaling pathways. This effect might also be useful in order to avoid unwanted side-effects and to drastically lower the costs for taxol-based therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis.

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Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef; Ferra, Bartłomiej; Hiszczyńska-Sawicka, Elżbieta; Lautenbach, Dariusz

2014-07-01

The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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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

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 ...

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

Science.gov (United States)

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

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.

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

Science.gov (United States)

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.

Science.gov (United States)

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.

Recruitment of human aquaporin 3 to internal membranes in the Plasmodium falciparum infected erythrocyte.

Science.gov (United States)

Bietz, Sven; Montilla, Irine; Külzer, Simone; Przyborski, Jude M; Lingelbach, Klaus

2009-09-01

The molecular mechanisms underlying the formation of the parasitophorous vacuolar membrane in Plasmodium falciparum infected erythrocytes are incompletely understood, and the protein composition of this membrane is still enigmatic. Although the differentiated mammalian erythrocyte lacks the machinery required for endocytosis, some reports have described a localisation of host cell membrane proteins at the parasitophorous vacuolar membrane. Aquaporin 3 is an abundant plasma membrane protein of various cells, including mammalian erythrocytes where it is found in distinct oligomeric states. Here we show that human aquaporin 3 is internalized into infected erythrocytes, presumably during or soon after invasion. It is integrated into the PVM where it is organized in novel oligomeric states which are not found in non-infected cells.

Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

Science.gov (United States)

Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

2007-05-01

Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

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...

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.

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.

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

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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.

Determination of 18 beta-glycyrrhetinic acid in biological fluids from humans and rats by solid-phase extraction and high-performance liquid chromatography.

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Hasler, F; Krapf, R; Brenneisen, R; Bourquin, D; Krähenbühl, S

1993-10-22

Methods have been developed and characterized allowing rapid isolation and quantification of 18 beta-glycyrrhetinic acid (GRA) in biological fluids from both humans and rats. Sample preparation includes extraction with urea-methanol for plasma samples, and solid-phase extraction (SPE) for urine and bile samples. Hydrolysis of GRA glucuronides in urine and bile was performed by treatment with beta-glucuronidase. MGRA, the 3-O-methyl derivative of GRA was synthesized as an internal standard resistant to hydrolysis. High-performance liquid chromatography (HPLC) was performed with an isocratic system using methanol-water-acetic acid (83:16.8:0.2, v/v/v) as solvent on a Lichrocart RP-18 column at 30 degrees C with ultraviolet detection. The methods allowed base line separation of GRA and MGRA from all biological fluids tested, with a detection limit of 0.15 mg/l. Validation of the methods included determination of recovery, accuracy and precision in plasma, bile and urine from humans and rats. The methods were further evaluated by investigating the pharmacokinetics of GRA in normal rats and in rats with a bile fistula. Following an intravenous dose of 10 mg/kg, the plasma concentration-time curve of GRA could be fitted to a one compartment model both in control and bile fistula rats. The elimination half life averaged 15.0 +/- 2.2 versus 16.8 +/- 2.4 min in control and bile fistula rats (difference not significant). Within 90 min following administration of GRA, urinary elimination of GRA and GRA glucuronides was less than 1% in both groups whereas biliary elimination averaged 51.3 +/- 3.1%. The results show that the methods developed allow pharmacokinetic studies of GRA in humans and rats.

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

Structures and immunolocalization of Na+, K+ -ATPase, Na+ /H+ exchanger 3 and vacuolar-type H+ -ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas.

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Uchiyama, M; Komiyama, M; Yoshizawa, H; Shimizu, N; Konno, N; Matsuda, K

2012-05-01

The structure and immunolocalization of the ion transporters Na(+) ,K(+) -ATPase (NKA), Na(+) /H(+) exchanger (NHE3) and vacuolar-type H(+) -ATPase (VHA) were examined in the gills of teleosts of the family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. These features were compared among the following species with different ecologies: the amphibious rockskipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimae and the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopic observations indicated that thick gill filaments were arranged close to each other and alternately on two hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC) and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. An immunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were located predominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses using serial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells (IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae, NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the base of the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E. yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located at the tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- and VHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. This study shows that the structure and distribution of ion transporters in the gills differ among the three blennid species, presumably reflecting their different ecologies. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the

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

Medidas higienistas adotadas no Patronato Agrícola Visconde da Graça (1923-34 - Pelotas/RS

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Magda de Abreu Vicente

2012-09-01

Full Text Available Esta pesquisa foi desenvolvida como parte de trabalho de mestrado junto ao Programa dePós-Graduação da Universidade Federal de Pelotas, Faculdade de Educação. Tem comoobjetivo analisar as normas higiênicas no Patronato Agrícola Visconde da Graça, noperíodo de 1923 até 1934, na cidade de Pelotas-RS e entender o enquadramento dasdiscussões higiênicas em voga no Brasil, a partir das notícias vinculadas no jornal DiárioPopular e das fichas dos alunos desta instituição. O texto, inicialmente, esboça asdiscussões brasileiras que elaboram o discurso higienista. Usa, para tal, referênciasbibliográficas que buscaram explicitar esta questão. Posteriormente, adentramos nestadiscussão em parâmetros locais, especificamente na cidade de Pelotas, através das notíciaspublicadas no jornal pelotense Diário Popular e também através de alguns pesquisadoresque aqui buscam entender as questões referentes às normas de higiene na cidade e naescola. Ao mesmo tempo analisamos como estas questões eram abordadas dentro doPatronato, através dos registros anotados nas fichas dos alunos que ali estiveram noperíodo estudado, pesquisadas no Acervo Escolar.

Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

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Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

2011-11-22

Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

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

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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.

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.

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

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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.

A systematic evaluation of three different cardiac T2-mapping sequences at 1.5 and 3T in healthy volunteers.

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Baeßler, Bettina; Schaarschmidt, Frank; Stehning, Christian; Schnackenburg, Bernhard; Maintz, David; Bunck, Alexander C

2015-11-01

Previous studies showed that myocardial T2 relaxation times measured by cardiac T2-mapping vary significantly depending on sequence and field strength. Therefore, a systematic comparison of different T2-mapping sequences and the establishment of dedicated T2 reference values is mandatory for diagnostic decision-making. Phantom experiments using gel probes with a range of different T1 and T2 times were performed on a clinical 1.5T and 3T scanner. In addition, 30 healthy volunteers were examined at 1.5 and 3T in immediate succession. In each examination, three different T2-mapping sequences were performed at three short-axis slices: Multi Echo Spin Echo (MESE), T2-prepared balanced SSFP (T2prep), and Gradient Spin Echo with and without fat saturation (GraSEFS/GraSE). Segmented T2-Maps were generated according to the AHA 16-segment model and statistical analysis was performed. Significant intra-individual differences between mean T2 times were observed for all sequences. In general, T2prep resulted in lowest and GraSE in highest T2 times. A significant variation with field strength was observed for mean T2 in phantom as well as in vivo, with higher T2 values at 1.5T compared to 3T, regardless of the sequence used. Segmental T2 values for each sequence at 1.5 and 3T are presented. Despite a careful selection of sequence parameters and volunteers, significant variations of the measured T2 values were observed between field strengths, MR sequences and myocardial segments. Therefore, we present segmental T2 values for each sequence at 1.5 and 3T with the inherent potential to serve as reference values for future studies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A Tonoplast P3B-ATPase Mediates Fusion of Two Types of Vacuoles in Petal Cells

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Marianna Faraco

2017-06-01

Full Text Available It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.

Fe(III) and Fe(II) ions different effects on Enterococcus hirae cell growth and membrane-associated ATPase activity

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Vardanyan, Zaruhi [Department of Biophysics of the Biology Faculty, Yerevan State University, 1 A. Manoukian Str., 0025 Yerevan (Armenia); Trchounian, Armen, E-mail: trchounian@ysu.am [Department of Biophysics of the Biology Faculty, Yerevan State University, 1 A. Manoukian Str., 0025 Yerevan (Armenia)

2012-01-06

Highlights: Black-Right-Pointing-Pointer Fe{sup 3+} stimulates but Fe{sup 2+} suppresses Enterococcus hirae wild-type and atpD mutant growth. Black-Right-Pointing-Pointer Fe ions change oxidation-reduction potential drop during cell growth. Black-Right-Pointing-Pointer Fe{sup 3+} and Fe{sup 2+} have opposite effects on a membrane-associated ATPase activity. Black-Right-Pointing-Pointer These effects are either in the presence of F{sub 0}F{sub 1} inhibitor or non-functional F{sub 0}F{sub 1}. Black-Right-Pointing-Pointer Fe ions decrease protons and coupled potassium ions fluxes across the membrane. -- Abstract: Enterococcus hirae is able to grow under anaerobic conditions during glucose fermentation (pH 8.0) which is accompanied by acidification of the medium and drop in its oxidation-reduction potential (E{sub h}) from positive values to negative ones (down to {approx}-200 mV). In this study, iron (III) ions (Fe{sup 3+}) have been shown to affect bacterial growth in a concentration-dependent manner (within the range of 0.05-2 mM) by decreasing lag phase duration and increasing specific growth rate. While iron(II) ions (Fe{sup 2+}) had opposite effects which were reflected by suppressing bacterial growth. These ions also affected the changes in E{sub h} values during bacterial growth. It was revealed that ATPase activity with and without N,N Prime -dicyclohexylcarbodiimide (DCCD), an inhibitor of the F{sub 0}F{sub 1}-ATPase, increased in the presence of even low Fe{sup 3+} concentration (0.05 mM) but decreased in the presence of Fe{sup 2+}. It was established that Fe{sup 3+} and Fe{sup 2+} both significantly inhibited the proton-potassium exchange of bacteria, but stronger effects were in the case of Fe{sup 2+} with DCCD. Such results were observed with both wild-type ATCC9790 and atpD mutant (with defective F{sub 0}F{sub 1}) MS116 strains but they were different with Fe{sup 3+} and Fe{sup 2+}. It is suggested that the effects of Fe{sup 3+} might be due to

Characterization of acute undifferentiated leukemia by combined analysis of plasma membrane-associated gamma-glutamyltranspeptidase and soluble terminal transferase.

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Heumann, D; Losa, G; Barras, C; Morell, A; von Fliedner, V

1985-08-01

gamma-Glutamyltranspeptidase (gamma-GT) is a plasma membrane-associated enzyme present in blasts of certain acute leukemias. We analyzed 90 cases of undifferentiated and differentiated acute leukemias for gamma-GT, using a colorimetric assay. Blasts of all patients with common acute lymphoblastic leukemia (ALL) and T-ALL were negative for gamma-GT (less than 5 units). In contrast, gamma-GT was significantly elevated in acute myeloblastic or monoblastic leukemia blasts (P less than .001). In 16 cases of acute undifferentiated leukemia (AUL) studied, the levels of gamma-GT ranged from 0 to 93 units; in eight cases, gamma-GT was positive (greater than 5 units), and six of these had 2% to 5% Sudan black-positive leukemic cells in the blast-enriched suspension. Combined gamma-GT/TdT analysis revealed that both enzyme markers were mutually exclusive in 75% of AUL cases, suggesting that gamma-GT+/TdT-blasts are of nonlymphoid origin, and gamma-GT-/TdT+ blasts are of lymphoid origin. Two cases were devoid of both enzyme activities and could represent truly undifferentiated leukemia. Thus, combined gamma-GT/TdT analysis underlines the heterogeneity of AUL and appears to be useful in defining the lineage commitment of undifferentiated leukemic blasts.

Mutations in Encephalomyocarditis Virus 3A Protein Uncouple the Dependency of Genome Replication on Host Factors Phosphatidylinositol 4-Kinase IIIα and Oxysterol-Binding Protein

NARCIS (Netherlands)

Dorobantu, Cristina M|info:eu-repo/dai/nl/372622283; Albulescu, Lucian|info:eu-repo/dai/nl/369492382; Lyoo, Heyrhyoung|info:eu-repo/dai/nl/412352931; van Kampen, Mirjam; De Francesco, Raffaele; Lohmann, Volker; Harak, Christian; van der Schaar, Hilde M|info:eu-repo/dai/nl/318007568; Strating, Jeroen R P M|info:eu-repo/dai/nl/298979594; Gorbalenya, Alexander E; van Kuppeveld, Frank J M|info:eu-repo/dai/nl/156614723

2016-01-01

Positive-strand RNA [(+)RNA] viruses are true masters of reprogramming host lipid trafficking and synthesis to support virus genome replication. Via their membrane-associated 3A protein, picornaviruses of the genus Enterovirus (e.g., poliovirus, coxsackievirus, and rhinovirus) subvert Golgi

Expression of FlHMA3, a P1B2-ATPase from Festulolium loliaceum, correlates with response to cadmium stress.

Science.gov (United States)

Guo, Qiang; Meng, Lin; Humphreys, Mike W; Scullion, John; Mur, Luis A J

2017-03-01

Heavy metal ATPase 3 (HMA3), a P 1B2 -ATPase, is a key tonoplast transporter involved in mediating the vacuolar sequestration of cadmium (Cd) to detoxify the intake of this element by plants. HMA3 expression in response to Cd stress has not been previously examined in the grass hybrid species Festulolium loliaceum (Huds.) P. Fourn. In this study, FlHMA3 isolated from F. loliaceum was found to comprise 833 amino acid residues with 77% homology to the rice OsHMA3. Transient expression of FlHMA3 fused to enhanced green fluorescent protein in Arabidopsis protoplasts suggested its localization to vacuolar membranes. Quantitative real-time RT-PCR analysis of F. loliaceum revealed that FlHMA3 is expressed predominantly within roots and up-regulated by excess Cd. Over the 168 h treatment, Cd content of F. loliaceum roots was significantly higher than that of shoots, regardless of external CdCl 2 concentrations. A significant positive correlation was found between FlHMA3 expression and Cd accumulation in roots of F. loliaceum seedlings subjected to 10-100 mg L -1 CdCl 2 for 168 h or, in a separate experiment, to 25 or 100 mg L -1 CdCl 2 for the same duration. These findings provide evidence that FlHMA3 encodes a vacuolar P 1B2 -ATPase that may play an important role in Cd 2+ sequestration into root cell vacuoles, thereby limiting the entry of Cd 2+ into the cytoplasm and reducing Cd 2+ toxicity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

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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

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.

Enhanced Salt Tolerance Conferred by the Complete 2.3 kb cDNA of the Rice Vacuolar Na(+)/H(+) Antiporter Gene Compared to 1.9 kb Coding Region with 5' UTR in Transgenic Lines of Rice.

Science.gov (United States)

Amin, U S M; Biswas, Sudip; Elias, Sabrina M; Razzaque, Samsad; Haque, Taslima; Malo, Richard; Seraj, Zeba I

2016-01-01

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na(+)/H(+) antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions (UTRs) of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5' UTR with CDS and the other of 2.3 kb, including 5' UTR, CDS, and the 3' UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5' and 3' UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3' UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P kb > 1.9 kb > and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance.

Determining the Pollinizer for Pecan Cultivars

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Fereidoon Ajamgard

2017-05-01

Full Text Available This study was conducted to determine the best pollinizer for five selected pecan cultivars in southwest of Iran at Safiabad Agricultural Research Center in 2014-2015. The cultivars included: 'GraTex', '10J', 'Wichita 6J', 'GraKing', 'Choctaw' as pollinated cultivars (♀ and 'GraTex', 'Peruque', 'Comanche 4M', '10J', 'Wichita 6J', 'Mohawk', 'Mahan', 'Stuart 2J', '3J', 'Stuart 4J', 'GraKing', 'Choctaw', 'Apache', '6M', 'Wichita 7J' and 'Comanche 5M.' as pollinizer cultivars (♂. In the first step, a pollination chart of cultivars was determined in two years. The pollination chart of cultivars showed that all the cultivars investigated during this study were dichogamous and also protogynous except for the 'Peruque'. ‘GraKing’ had the longest duration of shedding pollen. Pollination chart showed that 'Peruque', ‘GraKing’, and 'Stuart 2J' had flowering overlap with the selected cultivars. Pollen germination test showed that the germination ability was different among the cultivars. It was 45% for 'GraKing' and 35% for 'Peruque', which were both recommended as pollinizers in this study. '6M', 'GraTex' and 'Stuart 4J' cultivars had the highest pollen germination percentage of 65%, 60% and 60%, respectively. The results of controlled pollinationtest showed that different pollen sources had no significant effect on nuts per cluster but self-pollinated all of the cultivars significantly reduced fruit set in first and second years. Based on the present research, pollination in pecan orchard was necessary for adequate yield. Also, 'Peruque', 'GraKing' and 'Stuart 2J' were the best pollinizers for five selected cultivars in southwest of Iran.

Genotyping of polymorphic effectors of Toxoplasma gondii isolates from China

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Weisheng Cheng

2017-11-01

Full Text Available Abstract Background Toxoplasma gondii is an opportunistic protozoan apicomplexan and obligate intracellular parasite that infects a wide range of animals and humans. Rhoptry proteins 5 (ROP5, ROP16, ROP18 and dense granules 15 (GRA15 are the important effectors secreted by T. gondii which link to the strain virulence for mice and modulate the host’s response to the parasite. Little has been known about these molecules as well as GRA3 in type Chinese 1 strains that show polymorphism among strains of archetypical genotypes. This study examined the genetic diversity of these effectors and its correlated virulence in mice among T. gondii isolates from China. Results Twenty-one isolates from stray cats were detected, of which 15 belong to Chinese 1, and 6 to ToxoDB #205. Wh6 isolate, a Chinese 1 strain, has an avirulent phenotype. PCR-RFLP results of ROP5 and ROP18 presented few variations among the strains. Genotyping of GRA15 and ROP16 revealed that all the strains belong to type II allele except Xz7 which carries type I allele. ROP16 amino acid alignment at 503 locus demonstrated that 17 isolates are featured as type I or type III (ROP16I/III, and the other 4 as type II (ROP16II. The strains investigated may be divided into four groups based on GRA3 amino acid alignment, and all isolates of type Chinese 1 belong to the μ-1 allele except Wh6 which is identical to type II strain. Conclusions PCR-RFLP and sequence alignment analyses of ROP5, ROP16, ROP18, GRA3, and GRA15 in T. gondii revealed that strains with the same genotype may have variations in some of their key genes. GRA3 variation exhibited by Wh6 strain may be associated with the difference in phenotype and pathogenesis.

NCBI nr-aa BLAST: CBRC-DMEL-04-0042 [SEVENS

Lifescience Database Archive (English)

Full Text Available ike protein [Geobacter uraniumreducens Rf4] gb|ABQ26219.1| Uncharacterized membrane-associated protein-like protein [Geobacter uraniumreducens Rf4] YP_001230792.1 4.3 28% ... ...CBRC-DMEL-04-0042 ref|YP_001230792.1| Uncharacterized membrane-associated protein-l

Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

Science.gov (United States)

Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

2015-10-07

Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-gamma 1-binding phosphotyrosyl protein pp36/38

NARCIS (Netherlands)

Fukazawa, T.; Reedquist, K. A.; Panchamoorthy, G.; Soltoff, S.; Trub, T.; Druker, B.; Cantley, L.; Shoelson, S. E.; Band, H.

1995-01-01

Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of

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

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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.

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

reduced the clonogenic survival of MCF-7 cells from 0.70 to 0.51 following 2 Gy (p=0.01) and from 0.27 to 0.17 following 4 Gy (p<0.01). Concanamycin (2 pM) reduced the clonogenic survival following exposure to 3 Gy from 0.45 to 0.28 (p=0.015). LoVo cells exhibited a surviving fraction of 0.42 after 3 Gy, versus 0.21 with bafilomycin (p<0.01). Conclusion: These results suggest that formation of acidic vesicular organelles after irradiation serves to protect cells against radiation damage, and that interference with their acidification can decrease clonogenic cell survival post-radiation. Low concentrations of v-H+-ATPase inhibitors such as bafilomycin A1 and concanamycin sensitize cancer cells to radiation without affecting non-irradiated cells. The vacuolar-proton-ATPase can therefore provide a new target for modulation of the radiosensitivity of human epithelial cancer cells

Structural studies of alternative oxidase (AOX) from moniliophthora perniciosa, the causal agent of witches' broom disease in cacao: a membrane-associated protein

International Nuclear Information System (INIS)

Oliveira, J.F.; Prado, P.F.V.; Tiezzi, H.O.; Dias, S.M.G.; Ambrosio, A.L.B.; Thomazella, D.P.T.; Teixeira, P.J.P.L.; Pereira, G.A.G.

2012-01-01

Full text: Alternative oxidase (AOX) is a protein attached to the inner mitochondrial membrane that receives electrons directly from reduced ubiquinone and catalyzes the reduction of oxygen to water. AOX is a non-proton motive terminal quinol oxidase that enables cell respiration to continue even in the presence of inhibitors targeting the complexes of the respiratory chain. This protein is present in higher plants, pathogenic fungi and some parasites. The structural characterization of AOX becomes interesting due to its potential as a fungicide target. AOX is predicted to be a monotopic interfacial membrane protein interacting with a single leaflet of the lipid bilayer, rather than transmembrane. Amino acid sequence analysis reveals the presence of two conserved glutamate-histidine motifs, identifying it as a member of the diiron carboxylate protein family. The AOX model is defined by two pairs of helices forming a four helix bundle and an additional hydrophobic connecting sequence between the two helical pairs is proposed to act as the membrane anchoring region. In this work we aim at production, purification and crystallization of the AOX protein from M. perniciosa for further structural studies of this membrane-associated protein, by X-ray protein crystallography (author)

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.

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.

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

Science.gov (United States)

Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

2014-08-08

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Structural studies of alternative oxidase (AOX) from moniliophthora perniciosa, the causal agent of witches' broom disease in cacao: a membrane-associated protein

Energy Technology Data Exchange (ETDEWEB)

Oliveira, J.F.; Prado, P.F.V.; Tiezzi, H.O.; Dias, S.M.G.; Ambrosio, A.L.B. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Thomazella, D.P.T.; Teixeira, P.J.P.L.; Pereira, G.A.G. [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

2012-07-01

Full text: Alternative oxidase (AOX) is a protein attached to the inner mitochondrial membrane that receives electrons directly from reduced ubiquinone and catalyzes the reduction of oxygen to water. AOX is a non-proton motive terminal quinol oxidase that enables cell respiration to continue even in the presence of inhibitors targeting the complexes of the respiratory chain. This protein is present in higher plants, pathogenic fungi and some parasites. The structural characterization of AOX becomes interesting due to its potential as a fungicide target. AOX is predicted to be a monotopic interfacial membrane protein interacting with a single leaflet of the lipid bilayer, rather than transmembrane. Amino acid sequence analysis reveals the presence of two conserved glutamate-histidine motifs, identifying it as a member of the diiron carboxylate protein family. The AOX model is defined by two pairs of helices forming a four helix bundle and an additional hydrophobic connecting sequence between the two helical pairs is proposed to act as the membrane anchoring region. In this work we aim at production, purification and crystallization of the AOX protein from M. perniciosa for further structural studies of this membrane-associated protein, by X-ray protein crystallography (author)

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.

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

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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.

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.

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.

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

Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Gimble, F S; Thorner, J

1993-10-15

The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

Specific expression of the vacuolar iron transporter, TgVit, causes iron accumulation in blue-colored inner bottom segments of various tulip petals.

Science.gov (United States)

Momonoi, Kazumi; Tsuji, Toshiaki; Kazuma, Kohei; Yoshida, Kumi

2012-01-01

Several flowers of Tulipa gesneriana exhibit a blue color in the bottom segments of the inner perianth. We have previously reported the inner-bottom tissue-specific iron accumulation and expression of the vacuolar iron transporter, TgVit1, in tulip cv. Murasakizuisho. To clarify whether the TgVit1-dependent iron accumulation and blue-color development in tulip petals are universal, we analyzed anthocyanin, its co-pigment components, iron contents and the expression of TgVit1 mRNA in 13 cultivars which show a blue color in the bottom segments of the inner perianth accompanying yellow- and white-colored inner-bottom petals. All of the blue bottom segments contained the same anthocyanin component, delphinidin 3-rutinoside. The flavonol composition varied with cultivar and tissue part. The major flavonol in the bottom segments of the inner perianth was rutin. The iron content in the upper part was less than that in the bottom segments of the inner perianth. The iron content in the yellow and white petals was higher in the bottom segment of the inner perianth than in the upper tissues. TgVit1 mRNA expression was apparent in all of the bottom tissues of the inner perianth. The result of a reproduction experiment by mixing the constituents suggests that the blue coloration in tulip petals is generally caused by iron complexation to delphinidin 3-rutinoside and that the iron complex is solubilized and stabilized by flavonol glycosides. TgVit1-dependent iron accumulation in the bottom segments of the inner perianth might be controlled by an unknown system that differentiated the upper parts and bottom segments of the inner perianth.

Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins.

Science.gov (United States)

Chen, Xi; Howe, Jörg; Andrä, Jörg; Rössle, Manfred; Richter, Walter; da Silva, Ana Paula Galvão; Krensky, Alan M; Clayberger, Carol; Brandenburg, Klaus

2007-10-01

To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.

Adsorption of various types of amino acids on the graphene and boron-nitride nano-sheet, a DFT-D3 study

International Nuclear Information System (INIS)

Zhiani, Rahele

2017-01-01

Graphical abstract: Dispersion interactions have key role on the adsorption of different amino acids on the graphene and BN-nanosheet surfaces. - Highlights: • The Arginine amino acid makes the most stable complexes with Gra and BN nano sheet. • Dispersion interactions have key role on the amino acid adsorption. • BN nano sheet makes more stable complexes with amino acids compare to the Gra. • Water as a solvent has important effect on these interactions. - Abstract: The binding properties of the adsorption of five different classes of amino acids, namely, alanine (Ala), arginine (Arg), asparagine (Asn), histidine (His) and cysteine (Cys) on the surface of the graphene (Gra) and the born-nitride (BN) nano-sheet structures were studied from molecular viewpoint using quantum mechanics methods. Density functional theory (DFT) and DFT-D3 calculations were carried out to investigate the electronic properties and the dispersion interaction of the amino acid/adsorbent complexes. Several parameters affecting the interactions between the amino acids and the adsorbent surfaces such as solvent effect, adsorption energy and separation distance were investigated. Findings show that Arg forms the most stable complexes with the graphene and the BN nano-sheet compare to the other amino acids used in this study. The observed frequency results which were related to the band gap energies were consistent with the above statement. Results exhibit that adsorption of the amino acids on the surface of the BN nano-sheet and the graphene accompanied with the release of the energy. Calculations show that there are no bonded interactions between the amino acids and adsorbent surfaces. The polarity of the BN nano-sheet provides the more affinity towards the amino acids. These results were proved by the quantum chemistry studies.

Adsorption of various types of amino acids on the graphene and boron-nitride nano-sheet, a DFT-D3 study

Energy Technology Data Exchange (ETDEWEB)

Zhiani, Rahele, E-mail: r_zhiani2006@yahoo.com

2017-07-01

Graphical abstract: Dispersion interactions have key role on the adsorption of different amino acids on the graphene and BN-nanosheet surfaces. - Highlights: • The Arginine amino acid makes the most stable complexes with Gra and BN nano sheet. • Dispersion interactions have key role on the amino acid adsorption. • BN nano sheet makes more stable complexes with amino acids compare to the Gra. • Water as a solvent has important effect on these interactions. - Abstract: The binding properties of the adsorption of five different classes of amino acids, namely, alanine (Ala), arginine (Arg), asparagine (Asn), histidine (His) and cysteine (Cys) on the surface of the graphene (Gra) and the born-nitride (BN) nano-sheet structures were studied from molecular viewpoint using quantum mechanics methods. Density functional theory (DFT) and DFT-D3 calculations were carried out to investigate the electronic properties and the dispersion interaction of the amino acid/adsorbent complexes. Several parameters affecting the interactions between the amino acids and the adsorbent surfaces such as solvent effect, adsorption energy and separation distance were investigated. Findings show that Arg forms the most stable complexes with the graphene and the BN nano-sheet compare to the other amino acids used in this study. The observed frequency results which were related to the band gap energies were consistent with the above statement. Results exhibit that adsorption of the amino acids on the surface of the BN nano-sheet and the graphene accompanied with the release of the energy. Calculations show that there are no bonded interactions between the amino acids and adsorbent surfaces. The polarity of the BN nano-sheet provides the more affinity towards the amino acids. These results were proved by the quantum chemistry studies.

A compreensão da gratidão e teoria da mente em crianças de 5 anos The understanding of gratitude and theory of mind in 5-year-olds

Directory of Open Access Journals (Sweden)

Lia Beatriz de Lucca Freitas

2012-01-01

Full Text Available Examinaram-se relações entre compreensão da gratidão (GRA e desenvolvimento de uma teoria da mente (TOM com 228 crianças norteamericanas (53% meninas de 5 anos. Testaram-se as seguintes hipóteses: (a não há diferença de sexo em GRA ou TOM, (b as crianças com melhor desempenho nas tarefas de TOM têm melhor GRA, (c TOM é condição necessária para GRA. Utilizaram-se três tarefas para avaliar TOM: consideração da perspectiva visual, crença falsa de primeira e de segunda ordem. Contaram-se às crianças duas histórias sobre gratidão. Avaliou-se GRA a partir de suas respostas a perguntas feitas após a leitura de cada história. As duas primeiras hipóteses foram confirmadas. A hipótese de que TOM seria condição necessária para GRA não encontrou suficiente suporte empírico.We examined relations between the understanding of gratitude (GRA and the development of a theory of mind (TOM. The study was done with 228 5-year-old North American children (53% female. We tested the following hypotheses: (a there are no sex differences in GRA or TOM, (b children who perform better on TOM tasks have better GRA, (c TOM is a necessary condition for GRA. We used three tasks to evaluate TOM: visual perspective taking, first-order false belief, and second-order false belief. The children were read two vignettes about gratitude. GRA was evaluated based on children's responses to questions asked after each vignette was read. The first two hypotheses were supported. The hypothesis that TOM would be a necessary condition for GRA did not receive sufficient empirical support.

Calcium Signals from the Vacuole

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Gerald Schönknecht

2013-10-01

Full Text Available The vacuole is by far the largest intracellular Ca2+ store in most plant cells. Here, the current knowledge about the molecular mechanisms of vacuolar Ca2+ release and Ca2+ uptake is summarized, and how different vacuolar Ca2+ channels and Ca2+ pumps may contribute to Ca2+ signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca2+ transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca2+ channels that could elicit cytosolic [Ca2+] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca2+ release, but the molecular mechanism of this Ca2+ release still awaits elucidation. Some vacuolar Ca2+ channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca2+ signaling still has to be demonstrated. Ca2+ pumps in addition to establishing long-term Ca2+ homeostasis can shape cytosolic [Ca2+] transients by limiting their amplitude and duration, and may thus affect Ca2+ signaling.

Positional isomerism-driven two 3D pillar-layered metal-organic frameworks: Syntheses, topological structures and photoluminescence properties

Energy Technology Data Exchange (ETDEWEB)

Sun, Yayong; Zhao, Siwei; Ma, Haoran; Han, Yi; Liu, Kang; Wang, Lei, E-mail: inorchemwl@126.com

2016-06-15

Two novel three-dimensional (3D) pillar-layered metal-organic frameworks (MOFs), namely [Zn{sub 2}(μ{sub 2}-OH)(boaba)(1,4-bmimb)]{sub n} (1) and {[Zn_5K_2(μ_2-H_2O)_2(boaba)_4(1,2-bmimb)_2(H_2O)_2]·H_2O}{sub n} (2), were prepared by hydrothermal reactions (H{sub 3}boaba=3,5-bis-oxyacetate-benzoic acid; 1,4-bmimb=1,4-bis((2-methyl-1H-imidazol-1-yl)methyl)benzene; 1,2-bmimb =1,2-bis((2-methyl-1H-imidazol-1-yl)methyl)benzene). Notably, 1 exhibits a (3,5)-connected binodal (6{sup 3})(6{sup 9}·8)-gra net with binuclear [Zn{sub 2}(μ{sub 2}-OH)(COO)]{sup 2+} clusters, while 2 shows a novel (4,4,5,9)-connected 4-nodal net constructed from the unique Zn(II)-K(I) heterometal rod-like substructures. The results indicate that the disposition of the 2-methylimidazolyl groups of bis(imidazole) ligands have a significant effect on structural diversity. Moreover, the photoluminescence properties of 1 and 2 have been investigated. - Graphical abstract: Two novel 3D pillar-layered metal-organic coordination networks with aromatic multicarboxylate anion and bis(imidazole) ligands have been synthesized and structurally characterized. Display Omitted - Highlights: • It is rarely reported that metal-organic frameworks prepared with 3,5-bis-oxyacetate-benzoic acid. • Two metal-organic frameworks based on positional isomeric ligands were synthesized and structurally characterized. • Compond 1 displays unique (3,5)-connected binodal gra topology. • Compound 2 exhibits (4,4,5,9)-connected 4-nodal topology based on the Zn(II)-K(I) heterometal rod-like substructures. • The photoluminescence properties of compound 1 and 2 have been investigated.

Alternative expression of vacuolar iron transporter and ferritin genes leads to blue/purple coloration of flowers in tulip cv. 'Murasakizuisho'.

Science.gov (United States)

Shoji, Kazuaki; Momonoi, Kazumi; Tsuji, Tosiaki

2010-02-01

Flowers of tulip cv. 'Murasakizuisho' have a purple perianth except for the bottom region, which is blue in color even though it has the same anthocyanin, delphinidin 3-O-rutinoside, as the entire perianth. The development of the blue coloration in the perianth bottom is due to complexation by anthocyanin, flavonol and iron (Fe), as well as a vacuolar iron transporter, TgVit1. Although transient expression of TgVit1 in the purple cells led to a color change to light blue, the coloration of the transformed cells did not coincide with the dark blue color of the cells of the perianth bottom. We thought that another factor is required for the blue coloration of the cells of perianth bottom. To examine the effect of ferritin (FER), an Fe storage protein, on blue color development, we cloned an FER gene (TgFER1) and performed expression analyses. TgFER1 transcripts were found in the cells located in the upper region of the petals along with purple color development by anthocyanin and were not found in the blue cells of the perianth bottom. This gene expression is in contrast to that of TgVit1, expressed only in the cells of the perianth bottom. Co-expression of TgVIT1 and TgFER-RNAi, constructed for suppressing endogenous TgFER1 by RNA interference (RNAi), changed the purple petal cells to a dark blue color similar to that of the natural perianth bottom. These results strongly suggest that TgVit1 expression and TgFER1 suppression are critical for the development of blue color in the perianth bottom.

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

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

Science.gov (United States)

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.

High-Gradient Test of a 3 GHz Single-Cell Cavity

CERN Document Server

Verdú-Andrés, S; Bonomi, R; Degiovanni, A; Garlasché, M; Garonna, A; Mellace, C; Pearce, P; S. Verdú-Andrés; Wegner, R

2010-01-01

Pro­ton and car­bon ion beams pre­sent ad­van­ta­geous depth-dose dis­tri­bu­tions with re­spect to X-rays. Car­bon ions allow a bet­ter con­trol of "ra­diore­sis­tant" tu­mours due to their high­er bi­o­log­i­cal re­sponse. For deep-seat­ed tu­mours pro­ton and car­bon ion beams of some nA and en­er­gies of about 200 MeV and 400 MeV/u re­spec­tive­ly are need­ed. For these ap­pli­ca­tions TERA pro­posed the "cy­clinac": a high-fre­quen­cy linac which boosts the hadrons ac­cel­er­at­ed by a cy­clotron. The di­men­sions of the com­plex can be re­duced if high­er ac­cel­er­at­ing gra­di­ents are achieved in the linac. To test the max­i­mum achiev­able fields, a 3 GHz cav­i­ty has been built by TERA. The 19 mm-long cell is fore­seen to be ex­cit­ed at 200 Hz by 3 us RF puls­es and should reach a 40 MV/m ac­cel­er­at­ing gra­di­ent, which cor­re­sponds to a peak sur­face elec­tric field Es of 260 MV/m. In a first high-pow­er test per­for...

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

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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.

Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity.

Directory of Open Access Journals (Sweden)

Barbara A Fox

2016-07-01

Full Text Available Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP and dense granule (GRA proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately

Physiological effects of the form of nitrogen on corn root tips: a 31P nuclear magnetic resonance study

International Nuclear Information System (INIS)

Andrade, F.H.; Anderson, I.C.

1986-01-01

Physiological effects of different N forms (NO − 3 , NH + 4 , or a combination of both) on corn (Zea mays L.) root tips and leaves were studied by following 31 P signals with a nuclear magnetic resonance spectrometer. With root tips, both cytoplasmic and vacuolar pH could be measured, whereas with leaves, only vacuolar pH could be determined. The N treatments did not affect the cytoplasmic pH of corn root tips in contrast to proposals of previous workers. Leaf vacuolar pH was higher and root tip vacuolar pH lower with NO − 3 than with NH + 4 . Under anaerobic conditions, cytoplasmic pH was reduced because of lactic acid fermentation. Nitrate, an electron acceptor, delayed the acidification of the cytoplasm compartment because it represents an alternative way to reoxidize NADH. In conclusion, for the conditions of these experiments, the pH of the cytoplasm of corn root tips was not modified by the form of N absorbed; however, the pH of this compartment was affected by the form of N presented during development anaerobiosi. (author)

Generation Ratio Availability Assessment of Electrical Systems for Offshore Wind Farms

DEFF Research Database (Denmark)

Zhao, Menghua; Chen, Zhe; Blaabjerg, Frede

2007-01-01

An availability index, Generation Ratio Availability (GRA), is proposed to evaluate the electrical system of offshore wind farms (OWF). The GRA is the probability that at least a certain percent of wind power could be transferred to the grid system through the concerned electrical system. The GRA....... Comprehensive studies have been conducted to investigate the influence of the network design, component parameters, and wind-speed regimes on the GRA. The analysis presented in this paper is useful for both future wind farm planning and existing OWF evaluation....

Linguagens híbridas e publicidade: inovações na era do hipertexto 3.0

OpenAIRE

Gonçalves, Denise dos Santos; CEFET/MG

2013-01-01

O leitor ativo da era do Hipertexto 3.0 tem a sua escolha uma série de caminhos pelos quais navega de acordo com seu interesse pessoal (LANDOW, 2006), perpassando por textos constituídos por linguagens híbridas (SANTAELLA, 2005), que se cruzam graças às possibilidades geradas pela hipermídia. A publicidade lança mão dos recursos propiciados pela hipermídia para criar campanhas capazes de encantar o internauta e manter sua atenção durante o tempo necessário para conhecer os produtos. Analisamo...

Interactions of carbon nanotubes and/or graphene with manganese peroxidase during biodegradation of endocrine disruptors and triclosan.

Science.gov (United States)

Chen, Ming; Zeng, Guangming; Lai, Cui; Zhang, Chang; Xu, Piao; Yan, Min; Xiong, Weiping

2017-10-01

Molecular-level biodegradation processes of bisphenol A (BPA), nonylphenol (NP) and triclosan (TCS) mediated by manganese peroxidase (MnP) were investigated with and without single-walled carbon nanotube (SWCNT) and/or graphene (GRA). Although the incorporation of SWCNT, GRA or their combination (SWCNT+GRA) did not break up the complexes composed of manganese peroxidase (MnP) and these substrates, they had different effects on the native contacts between the substrates and MnP. GRA tended to decrease the overall stability of the binding between MnP and its substrates. SWCNT or SWCNT+GRA generally had a minor impact on the mean binding energy between MnP and its substrates. We detected some sensitive residues from MnP that were dramatically disturbed by the GRA, SWCNT or SWCNT+GRA. Nanomaterials changed the number and behavior of water molecules adjacent to both MnP and its substrates, which was not due to the destruction of H-bond network formed by sensitive regions and water molecules. The present results are useful for understanding the molecular basis of pollutant biodegradation affected by the nanomaterials in the environment, and are also helpful in assessing the risks of these materials to the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Oxidation Stability of Pig Liver Pâté with Increasing Levels of Natural Antioxidants (Grape and Tea

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Mirian Pateiro

2015-01-01

Full Text Available The present study investigated the effect of the addition of increasing levels of the natural antioxidants tea (TEA and grape seed extracts (GRA on the physiochemical and oxidative stability of refrigerated stored pig pâtés. In addition, a synthetic antioxidant and a control batch were used, thus a total of eight batches of liver pâté were prepared: CON, BHT, TEA (TEA50, TEA200 and TEA1000 and GRA (GRA50, GRA200 and GRA1000. Pâté samples were analyzed following 0, 4, 8 and 24 weeks of storage. Color parameters were affected by storage period and level of antioxidant extract. Samples with TEA200 and GRA1000 levels of extracts showed lower total color difference between 0 and 24 weeks. At the end of storage period, the lower TBARs values were obtained in samples with the highest concentration on natural extract. Overall, the evolution of volatile compounds showed an increase in those ones that arise from the lipid oxidation and samples with TEA1000 extract showed the lowest values.

Metabolic Response of Maize Roots to Hyperosmotic Shock 1

Science.gov (United States)

Spickett, Corinne M.; Smirnoff, Nicholas; Ratcliffe, R. George

1992-01-01

31P nuclear magnetic resonance spectroscopy was used to study the response of maize (Zea mays L.) root tips to hyperosmotic shock. The aim was to identify changes in metabolism that might be relevant to the perception of low soil water potential and the subsequent adaptation of the tissue to these conditions. Osmotic shock was found to result in two different types of response: changes in metabolite levels and changes in intracellular pH. The most notable metabolic changes, which were produced by all the osmotica tested, were increases in phosphocholine and vacuolar phosphate, with a transient increase in cytoplasmic phosphate. It was observed that treatment with ionic and nonionic osmotica produced different effects on the concentrations of bioenergetically important metabolites. It is postulated that these changes are the result of hydrolysis of phosphatidylcholine and other membrane phospholipids, due to differential activation of specific membrane-associated phospholipases by changes in the surface tension of the plasmalemma. These events may be important in the detection of osmotic shock and subsequent acclimatization. A cytoplasmic alkalinization was also observed during hyperosmotic treatment, and this response, which is consistent with the activation of the plasmalemma H+-ATPase, together with the other metabolic changes, may suggest the existence of a complex and integrated mechanism of osmoregulation. PMID:16669012

ALTERNATE FOOD-CHAIN TRANSFER OF THE TOXIN LINKED TO AVIAN VACUOLAR MYELINOPATHY AND IMPLICATIONS FOR THE ENDANGERED FLORIDA SNAIL KITE (ROSTRHAMUS SOCIABILIS).

Science.gov (United States)

Dodd, Shelley R; Haynie, Rebecca S; Williams, Susan M; Wilde, Susan B

2016-04-28

Avian vacuolar myelinopathy (AVM) is a neurologic disease causing recurrent mortality of Bald Eagles ( Haliaeetus leucocephalus ) and American Coots ( Fulica americana ) at reservoirs and small impoundments in the southern US. Since 1994, AVM is considered the cause of death for over 170 Bald Eagles and thousands of American Coots and other species of wild birds. Previous studies link the disease to an uncharacterized toxin produced by a recently described cyanobacterium, Aetokthonos hydrillicola gen. et sp. nov. that grows epiphytically on submerged aquatic vegetation (SAV). The toxin accumulates, likely in the gastrointestinal tract of waterbirds that consume SAV, and birds of prey are exposed when feeding on the moribund waterbirds. Aetokthonos hydrillicola has been identified in all reservoirs where AVM deaths have occurred and was identified growing abundantly on an exotic SAV hydrilla ( Hydrilla verticillata ) in Lake Tohopekaliga (Toho) in central Florida. Toho supports a breeding population of a federally endangered raptor, the Florida Snail Kite ( Rostrhamus sociabilis ) and a dense infestation of an exotic herbivorous aquatic snail, the island applesnail ( Pomacea maculata ), a primary source of food for resident Snail Kites. We investigated the potential for transmission in a new food chain and, in laboratory feeding trials, confirmed that the AVM toxin was present in the hydrilla/A. hydrillicola matrix collected from Toho. Additionally, laboratory birds that were fed apple snails feeding on hydrilla/A. hydrillicola material from a confirmed AVM site displayed clinical signs (3/5), and all five developed brain lesions unique to AVM. This documentation of AVM toxin in central Florida and the demonstration of AVM toxin transfer through invertebrates indicate a significant risk to the already diminished population of endangered Snail Kites.

Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

OpenAIRE

Preston, R A; Manolson, M F; Becherer, K; Weidenhammer, E; Kirkpatrick, D; Wright, R; Jones, E W

1991-01-01

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX...

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.

Toxicity of fatty acid 18:5n3 from Gymnodinium cf. mikimotoi: II. Intracellular pH and K+ uptake in isolated trout hepatocytes.

Science.gov (United States)

Fossat, B; Porthé-Nibelle, J; Sola, F; Masoni, A; Gentien, P; Bodennec, G

1999-01-01

Effects of octadecapentaenoic acid 18:5n3 and other related polyunsaturated fatty acids present in gymnodinium cf. mikimotoi were tested in isolated trout hepatocytes. These exotoxins decreased intracellular pH followed by a slow recovery to initial value and alkalinization of acidic compartments, suggesting an inhibition of vacuolar H(+)-ATPases. Moreover, addition of 18:5n3 to the extracellular medium induced a decrease of K+ uptake into hepatocytes as a result of Na,K-ATPase inhibition. However, high concentrations (10(-5)-10(-3) M) are necessary to induce these effects.

Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)

International Nuclear Information System (INIS)

Baldassare, J.J.; Henderson, P.A.; Fisher, G.J.

1989-01-01

The effects of thrombin and GTPγS on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [ 3 H]inositol-labeled membranes or with lipid vesicles containing either [ 3 H]phosphatidylinositol or [ 3 H]phosphatidylinositol 4,5-bisphosphate. GTPγS (1 μM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP 3 ), inositol bisphosphate (IP 2 ), or inositol phosphate (IP) from [ 3 H]inositol-labeled membranes. IP 2 and IP 3 , but not IP, from [ 3 H]inositol-labeled membranes were, however, stimulated 3-fold by GTPγS (1 μM) plus thrombin (1 unit/mL). A higher concentration of GTPγS (100 μM) alone also stimulated IP 2 and IP 3 , but not IP, release. In the presence of 1 mM calcium, release of IP 2 and IP 3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by platelet membrane associated PLC was also markedly enhanced by GTPγS (100 μM) or GTPγS (1 μM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP 2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTPγS (100 μM) or calcium (1 mM) dependent PIP 2 breakdown, while TPA inhibited GTPγS-dependent but not calcium-dependent phospholipase C activity

SwissProt search result: AK105135 [KOME

Lifescience Database Archive (English)

Full Text Available ulfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 3e-61 ...

Linking salinity stress tolerance with tissue-specific Na+ sequestration in wheat roots

Directory of Open Access Journals (Sweden)

Honghong eWu

2015-02-01

Full Text Available Salinity stress tolerance is a physiologically complex trait that is conferred by the large array of interacting mechanisms. Among these, vacuolar Na+ sequestration has always been considered as one of the key components differentiating between sensitive and tolerant species and genotypes. However, vacuolar Na+ sequestration has been rarely considered in the context of the tissue-specific expression and regulation of appropriate transporters contributing to Na+ removal from the cytosol. In this work, six bread wheat varieties contrasting in their salinity tolerance (three tolerant and three sensitive were used to understand the essentiality of vacuolar Na+ sequestration between functionally different root tissues, and link it with the overall salinity stress tolerance in this species. Roots of 4-d old wheat seedlings were treated with 100 mM NaCl for 3 days, and then Na+ distribution between cytosol and vacuole was quantified by CoroNa Green fluorescent dye imaging. Our major observations were as follows: 1 salinity stress tolerance correlated positively with vacuolar Na+ sequestration ability in the mature root zone but not in the root apex; 2 Contrary to expectations, cytosolic Na+ levels in root meristem were significantly higher in salt tolerant than sensitive group, while vacuolar Na+ levels showed an opposite trend. These results are interpreted as meristem cells playing a role of the salt sensor; 3 No significant difference in the vacuolar Na+ sequestration ability was found between sensitive and tolerant group in either transition or elongation zones; 4 The overall Na+ accumulation was highest in the elongation zone, suggesting its role in osmotic adjustment and turgor maintenance required to drive root expansion growth. Overall, the reported results suggest high tissue-specificity of Na+ uptake, signalling, and sequestration in wheat root. The implications of these findings for plant breeding for salinity stress tolerance are discussed.

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

2016-01-01

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

Evolutionarily Conserved 5 '-3 ' Exoribonuclease Xrn1 Accumulates at Plasma Membrane-Associated Eisosomes in Post-Diauxic Yeast

Czech Academy of Sciences Publication Activity Database

Groušl, Tomáš; Opekarová, Miroslava; Strádalová, Vendula; Hašek, Jiří; Malínský, Jan

2015-01-01

Roč. 10, č. 3 (2015) E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GAP302/11/0146; GA ČR GA15-10641S Institutional support: RVO:61388971 ; RVO:68378041 Keywords : MAMMALIAN STRESS GRANULES * EUKARYOTIC MESSENGER-RNAS * CYTOPLASMIC PROCESSING BODIES Subject RIV: EE - Microbiology, Virology; EA - Cell Biology (UEM-P) Impact factor: 3.057, year: 2015

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.

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.

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.

Evaluation of web-based, self-administered, graphical food frequency questionnaire.

Science.gov (United States)

Kristal, Alan R; Kolar, Ann S; Fisher, James L; Plascak, Jesse J; Stumbo, Phyllis J; Weiss, Rick; Paskett, Electra D

2014-04-01

Computer-administered food frequency questionnaires (FFQs) can address limitations inherent in paper questionnaires by allowing very complex skip patterns, portion size estimation based on food pictures, and real-time error checking. We evaluated a web-based FFQ, the Graphical Food Frequency System (GraFFS). Participants completed the GraFFS, six telephone-administered 24-hour dietary recalls over the next 12 weeks, followed by a second GraFFS. Participants were 40 men and 34 women, aged 18 to 69 years, living in the Columbus, OH, area. Intakes of energy, macronutrients, and 17 micronutrients/food components were estimated from the GraFFS and the mean of all recalls. Bias (second GraFFS minus recalls) was -9%, -5%, +4%, and -4% for energy and percentages of energy from fat, carbohydrate, and protein, respectively. De-attenuated, energy-adjusted correlations (intermethod reliability) between the recalls and the second GraFFS for fat, carbohydrate, protein, and alcohol were 0.82, 0.79, 0.67, and 0.90, respectively; for micronutrients/food components the median was 0.61 and ranged from 0.40 for zinc to 0.92 for beta carotene. The correlations between the two administrations of the GraFFS (test-retest reliability) for fat, carbohydrate, protein, and alcohol were 0.60, 0.63, 0.73, and 0.87, respectively; among micronutrients/food components the median was 0.67 and ranged from 0.49 for vitamin B-12 to 0.82 for fiber. The measurement characteristics of the GraFFS were at least as good as those reported for most paper FFQs, and its high intermethod reliability suggests that further development of computer-administered FFQs is warranted. Copyright © 2014 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.

The Group Reminiscence Approach Can Increase Self-Awareness of Memory Deficits and Evoke a Life Review in People With Mild Cognitive Impairment: The Kurihara Project Data.

Science.gov (United States)

Nakamura, Kei; Kasai, Mari; Nakai, Megumi; Nakatsuka, Masahiro; Meguro, Kenichi

2016-06-01

The group reminiscence approach (GRA) and reality orientation (RO) are common psychosocial interventions for patients with dementia. As a qualitative evaluation of the reminiscence approach in patients with dementia, the Patient Report Outcome (PRO) is useful. The purpose of this study was to investigate the effects of GRA-RO for participants with mild cognitive impairment (MCI) using the PRO. A cluster randomized controlled trial. Community-based study. Ninety-four patients with MCI (39 GRA-RO, 23 physical activity, and 32 cognitive training) described their impressions. Based on the database of the Kurihara Project, we retrospectively analyzed the participants' descriptions of their impressions as a PRO in the nonpharmacological interventions: GRA-RO, physical activity, and cognitive training. We categorized the descriptions according to the following 2 types: impression with content and reminiscence with life review. We assessed what they wrote regarding memory loss. The content on their life reviews was also a particular focus for the GRA-RO group. PRO. Compared with the physical activity and the clinical training groups, the GRA-RO patients described their reminiscence with life review and their own memory problems. There was no confusion of the order of events of their autobiographical memories. There was a significant time effect between the 2 family involvement groups in quality-of-life (QOL) scores, and the postintervention QOL scores were significantly better than preintervention. This study suggests that the GRA-RO in participants with MCI not only stimulates life review but also increases self-awareness of memory deficits without confusion of the order of events. Thus, the GRA-RO may improve self-esteem and develop self-awareness. Copyright © 2016 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.

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.

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.

MIL-STD-2411-1 Change 3. Notice Impacts to NAVAIR

Science.gov (United States)

2010-07-30

Macedonia ZX Macais Nguema Biyogo MA Madagascar VD Madeira Islands (PO) MI Malawi MY Malaysia MV Maldives ML Mali MT Malta IM Isle of Man (UK) MIL...and Niger) PON Pointe Noire 1948 (Congo) POS Porto Santo 1936 (Porto Santo, Madeira Islands) HIT Prov. S. Chilean (S. Chile, 53 S.) PRPM Prov. S...Asia (Southeast Asia, Singapore) POS SE Base (Porte Santo) (Porto Santo & Madeira Islands) GRA SW Base (Faial, Graciosa, Pico, Sao Jorge, and

Na+/K+-ATPase and vacuolar-type H+-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation.

Science.gov (United States)

Huang, Chun-Yen; Chao, Pei-Lin; Lin, Hui-Chen

2010-03-01

The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na(+)/K(+)-ATPase (NKA) and vacuolar-type H(+)-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na(+) concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model. Copyright 2009 Elsevier B.V. All rights reserved.

αS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form

Science.gov (United States)

2010-01-01

Background Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known. Results In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both αS1- and β-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature β-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature αS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of αS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of αS1-casein with membranes. Conclusions These experiments reveal for the first time the existence of a membrane-associated form of αS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that αS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway. PMID:20704729

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.)

A Toxoplasma gondii protein with homology to intracellular type Na+/H+ exchangers is important for osmoregulation and invasion

International Nuclear Information System (INIS)

Francia, Maria E.; Wicher, Sarah; Pace, Douglas A.; Sullivan, Jack; Moreno, Silvia N.J.; Arrizabalaga, Gustavo

2011-01-01

The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na + /H + exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca 2+ concentration [Ca 2+ ] i , and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

Membrane-associated signaling in human B-lymphoma lines

Energy Technology Data Exchange (ETDEWEB)

Tauzin, Sebastien; Ding, Heidrun; Burdevet, Dimitri [Department of Pathology and Immunology, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland); Borisch, Bettina [Department of Social and Preventive Medicine, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland); Hoessli, Daniel C., E-mail: danielhoessli@gmail.com [Department of Pathology and Immunology, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland)

2011-01-15

In B-non-Hodgkin lymphomas, Lyn and Cbp/PAG constitute the core of an oncogenic signalosome that captures the Phosphatidylinositol-3-kinase, the Spleen tyrosine kinase and the Signal transducer and activator of transcription-3 to generate pro-survival and proliferative signals. Lymphoma lines corresponding to follicular, mantle-cell and Burkitt-derived lymphomas display type-specific signalosome organizations that differentially activate PI3K, Syk and STAT3. In the follicular lymphoma line, PI3K, Syk and STAT3 were optimally activated upon association with the Lyn-Cbp/PAG signalosome, while in the Burkitt lymphoma-derived line, the association with Cbp/PAG and activation of PI3K were interfered with by the latent membrane proteins encoded by the Epstein-Barr virus. In the Jeko-1 mantle-cell line, a weak association of Syk with the Lyn-Cbp/PAG signalosome resulted in poor activation of Syk, but in those cells, as in the follicular and Burkitt-derived lines, efficient apoptosis induction by the Syk inhibitor R406 indicated that Syk is nonetheless an important prosurvival element and therefore a valuable therapeutic target. In all configurations described herein is the Lyn-Cbp/PAG signalosome independent of external signals and provides efficient means of activation for its associated lipid and protein kinases. In follicular and Burkitt-derived lines, Syk appears to be activated following binding to Cbp/PAG and no longer requires B-cell receptor-associated activation motifs for activation. Assessment of the different modalities of Lyn-Cbp/PAG signalosome organization could help in selecting the appropriate combination of kinase inhibitors to eliminate a particular type of lymphoma cells.

Complexes of membrane-associated gamma-tubulin with Fyn kinase and phosphoinositide 3-kinase in differentiating cells

Czech Academy of Sciences Publication Activity Database

Sulimenko, Vadym; Macůrek, Libor; Dráberová, Eduarda; Richterová, Věra; Sulimenko, Tetyana; Dráberová, Lubica; Marková, Vladimíra; Dráber, Pavel

2009-01-01

Roč. 276, č. 1 (2009), s. 253-253 ISSN 1742-464X. [FEBS Congress /34/. 04.07.2009-09.07.2009, Praha] R&D Projects: GA MŠk 1M0506; GA MŠk LC545; GA ČR GA204/05/2375; GA ČR GA304/04/1273 Institutional research plan: CEZ:AV0Z50520514 Keywords : detergent -resistant membrane * Fyn * PI3K gamma-tubulin Subject RIV: EB - Genetics ; Molecular Biology

Experimental feeding of Hydrilla verticillata colonized by stigonematales cyanobacteria induces vacuolar myelinopathy in painted turtles (Chrysemys picta.

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Albert D Mercurio

Full Text Available Vacuolar myelinopathy (VM is a neurologic disease primarily found in birds that occurs when wildlife ingest submerged aquatic vegetation colonized by an uncharacterized toxin-producing cyanobacterium (hereafter "UCB" for "uncharacterized cyanobacterium". Turtles are among the closest extant relatives of birds and many species directly and/or indirectly consume aquatic vegetation. However, it is unknown whether turtles can develop VM. We conducted a feeding trial to determine whether painted turtles (Chrysemys picta would develop VM after feeding on Hydrilla (Hydrilla verticillata, colonized by the UCB (Hydrilla is the most common "host" of UCB. We hypothesized turtles fed Hydrilla colonized by the UCB would exhibit neurologic impairment and vacuolation of nervous tissues, whereas turtles fed Hydrilla free of the UCB would not. The ability of Hydrilla colonized by the UCB to cause VM (hereafter, "toxicity" was verified by feeding it to domestic chickens (Gallus gallus domesticus or necropsy of field collected American coots (Fulica americana captured at the site of Hydrilla collections. We randomly assigned ten wild-caught turtles into toxic or non-toxic Hydrilla feeding groups and delivered the diets for up to 97 days. Between days 82 and 89, all turtles fed toxic Hydrilla displayed physical and/or neurologic impairment. Histologic examination of the brain and spinal cord revealed vacuolations in all treatment turtles. None of the control turtles exhibited neurologic impairment or had detectable brain or spinal cord vacuolations. This is the first evidence that freshwater turtles can become neurologically impaired and develop vacuolations after consuming toxic Hydrilla colonized with the UCB. The southeastern United States, where outbreaks of VM occur regularly and where vegetation colonized by the UCB is common, is also a global hotspot of freshwater turtle diversity. Our results suggest that further investigations into the effect of the

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.

Likelihood analysis of the next-to-minimal supergravity motivated model

International Nuclear Information System (INIS)

Balazs, Csaba; Carter, Daniel

2009-01-01

In anticipation of data from the Large Hadron Collider (LHC) and the potential discovery of supersymmetry, we calculate the odds of the next-to-minimal version of the popular supergravity motivated model (NmSuGra) being discovered at the LHC to be 4:3 (57%). We also demonstrate that viable regions of the NmSuGra parameter space outside the LHC reach can be covered by upgraded versions of dark matter direct detection experiments, such as super-CDMS, at 99% confidence level. Due to the similarities of the models, we expect very similar results for the constrained minimal supersymmetric standard model (CMSSM).

Concurrent approach for evolving compact decision rule sets

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Marmelstein, Robert E.; Hammack, Lonnie P.; Lamont, Gary B.

1999-02-01

The induction of decision rules from data is important to many disciplines, including artificial intelligence and pattern recognition. To improve the state of the art in this area, we introduced the genetic rule and classifier construction environment (GRaCCE). It was previously shown that GRaCCE consistently evolved decision rule sets from data, which were significantly more compact than those produced by other methods (such as decision tree algorithms). The primary disadvantage of GRaCCe, however, is its relatively poor run-time execution performance. In this paper, a concurrent version of the GRaCCE architecture is introduced, which improves the efficiency of the original algorithm. A prototype of the algorithm is tested on an in- house parallel processor configuration and the results are discussed.

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 grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis.

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Martins, Viviana; Carneiro, Filipa; Conde, Carlos; Sottomayor, Mariana; Gerós, Hernâni

2017-12-01

The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca 2+ , Na + and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca 2+ homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca 2+ , demonstrating a role in Ca 2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na + , Li + , Mn 2+ and Cu 2+ in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca 2+ , and Na + in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.

Regulation of Serum Response Factor and Adiponectin by PPARγ Agonist Docosahexaenoic Acid

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Clayton Johnson

2011-01-01

Full Text Available Recent studies indicate that significant health benefits involving the regulation of signaling proteins result from the consumption of omega-3 polyunsaturated fatty acids (ω-3 PUFAs. Serum response factor (SRF is involved in transcriptional regulation of muscle growth and differentiation. SRF levels are increased in the aging heart muscle. It has not been examined whether SRF is made by adipocytes and whether SRF secretion by adipocytes is modulated by PPARγ agonist DHA. Adiponectin is made exclusively by adipocytes. We and others have previously reported that PUFAs such as DHA increase adiponectin levels and secretion in adipocytes. Here we show that DHA downregulates SRF with a simultaneous upregulation of adiponectin and that both these responses are reversible by PPARγ antagonist. Furthermore, there appears to be a direct relationship between DHA exposure and increased levels of membrane-associated high-density adiponectin, as well as lower levels of membrane-associated SRF. Thus, we find that the levels of SRF and adiponectin are inversely related in response to treatment with PPARγ agonist DHA. Decreased levels of SRF along with increase in membrane-associated adiponectin could in part mediate the health benefits of DHA.

Palladium nanoparticles anchored on graphene nanosheets: Methanol, ethanol oxidation reactions and their kinetic studies

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Nagaraju, D.H., E-mail: dhnagu@gmail.com [Department of Mechanical Engineering, 117 576 (Singapore); Materials Science and Engineering, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900 (Saudi Arabia); Devaraj, S. [Department of Mechanical Engineering, 117 576 (Singapore); School of Chemical and Biotechnology, SASTRA University, Thanjavur, 613 401 (India); Balaya, P., E-mail: mpepb@nus.edu.sg [Department of Mechanical Engineering, 117 576 (Singapore); Engineering Science Program, National University of Singapore, 117 576 (Singapore)

2014-12-15

Highlights: • Palladium nanoparticles decorated graphene is synthesized in a single step. • Electro-catalytic activity of Gra/Pd toward alcohol oxidation is evaluated. • 1:1 Gra/Pd exhibits good electro-catalytic activity and efficient electron transfer. - Abstract: Palladium nanoparticles decorated graphene (Gra/Pd nanocomposite) was synthesized by simultaneous chemical reduction of graphene oxide and palladium salt in a single step. The negatively charged graphene oxide (GO) facilitates uniform distribution of Pd{sup 2+} ions onto its surface. The subsequent reduction by hydrazine hydrate provides well dispersed Pd nanoparticles decorated graphene. Different amount of Pd nanoparticles on graphene was synthesized by changing the volume to weight ratio of GO to PdCl{sub 2}. X-ray diffraction studies showed FCC lattice of Pd with predominant (1 1 1) plane. SEM and TEM studies revealed that thin graphene nanosheets are decorated by Pd nanoparticles. Raman spectroscopic studies revealed the presence of graphene nanosheets. The electro-catalytic activity of Gra/Pd nanocomposites toward methanol and ethanol oxidation in alkaline medium was evaluated by cyclic voltammetric studies. 1:1 Gra/Pd nanocomposite exhibited good electro-catalytic activity and efficient electron transfer. The kinetics of electron transfer was studied using chronoamperometry. Improved electro-catalytic activity of 1:1 Gra/Pd nanocomposite toward alcohol oxidation makes it as a potential anode for the alcohol fuel cells.

Palladium nanoparticles anchored on graphene nanosheets: Methanol, ethanol oxidation reactions and their kinetic studies

International Nuclear Information System (INIS)

Nagaraju, D.H.; Devaraj, S.; Balaya, P.

2014-01-01

Highlights: • Palladium nanoparticles decorated graphene is synthesized in a single step. • Electro-catalytic activity of Gra/Pd toward alcohol oxidation is evaluated. • 1:1 Gra/Pd exhibits good electro-catalytic activity and efficient electron transfer. - Abstract: Palladium nanoparticles decorated graphene (Gra/Pd nanocomposite) was synthesized by simultaneous chemical reduction of graphene oxide and palladium salt in a single step. The negatively charged graphene oxide (GO) facilitates uniform distribution of Pd 2+ ions onto its surface. The subsequent reduction by hydrazine hydrate provides well dispersed Pd nanoparticles decorated graphene. Different amount of Pd nanoparticles on graphene was synthesized by changing the volume to weight ratio of GO to PdCl 2 . X-ray diffraction studies showed FCC lattice of Pd with predominant (1 1 1) plane. SEM and TEM studies revealed that thin graphene nanosheets are decorated by Pd nanoparticles. Raman spectroscopic studies revealed the presence of graphene nanosheets. The electro-catalytic activity of Gra/Pd nanocomposites toward methanol and ethanol oxidation in alkaline medium was evaluated by cyclic voltammetric studies. 1:1 Gra/Pd nanocomposite exhibited good electro-catalytic activity and efficient electron transfer. The kinetics of electron transfer was studied using chronoamperometry. Improved electro-catalytic activity of 1:1 Gra/Pd nanocomposite toward alcohol oxidation makes it as a potential anode for the alcohol fuel cells

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.

Control of nausea with palonosetron versus granisetron, both combined with dexamethasone, in patients receiving cisplatin- or anthracycline plus cyclophosphamide-based regimens.

Science.gov (United States)

Kubota, Kaoru; Saito, Mitsue; Aogi, Kenjiro; Sekine, Ikuo; Yoshizawa, Hirohisa; Yanagita, Yasuhiro; Sakai, Hiroshi; Inoue, Kenichi; Kitagawa, Chiyoe; Ogura, Takashi

2016-09-01

In a comparative phase 3 study involving 1114 Japanese patients receiving highly emetogenic chemotherapy (HEC), palonosetron (PALO) was found to be superior to granisetron (GRA) for the prophylaxis of chemotherapy-induced nausea and vomiting (CINV) in the delayed phase. This post hoc analysis of the phase 3 study evaluated the efficacy of PALO for the control of nausea. The proportion of patients without nausea was assessed at 24-h intervals during the acute phase (0-24 h), delayed phase (24-120 h), and overall (0-120 h). No nausea rates were also evaluated by sex, type of chemotherapy (cisplatin or doxorubicin/epirubicin plus cyclophosphamide [AC/EC]), and age (<55 vs. ≥55 years). Nausea severity was categorized using a 4-point Likert scale (0 = no nausea to 3 = severe nausea). The proportion of patients without nausea was significantly higher in the PALO arm than in the GRA arm in the delayed phase (37.8 % vs. 27.2 %; p = 0.002) and overall (31.9 % vs. 25.0 %; p = 0.0117). When analyzed by stratification factors, the proportion of patients without nausea was significantly higher in the PALO arm in the delayed phase and overall in patients who were female, younger, or treated with cisplatin and in the delayed phase in patients who were older or treated with doxorubicin or epirubicin plus cyclophosphamide (all p < 0.05). PALO was more effective than GRA in prophylaxis of HEC-induced nausea in the delayed phase and overall. In addition, PALO was more effective than GRA in young and female patients, who are at high risk of CINV, both in the delayed phase and overall.

Effects of gramicidin-A on the adsorption of phospholipids to the air–water interface

Science.gov (United States)

Biswas, Samares C.; Rananavare, Shankar B.; Hall, Stephen B.

2012-01-01

Prior studies suggest that the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of the lipids in pulmonary surfactant to an air–water interface by stabilizing a negatively curved rate-limiting structure that is intermediate between bilayer vesicles and the surface film. This model predicts that other peptides capable of stabilizing negative curvature should also promote lipid adsorption. Previous reports have shown that under appropriate conditions, gramicidin-A (GrA) induces dioleoyl phosphatidylcholine (DOPC), but not dimyristoyl phosphatidylcholine (DMPC), to form the negatively curved hexagonal-II (HII) phase. The studies reported here determined if GrA would produce the same effects on adsorption of DMPC and DOPC that the hydrophobic surfactant proteins have on the surfactant lipids. Small angle X-ray scattering and 31P-nuclear magnetic resonance confirmed that at the particular conditions used to study adsorption, GrA induced DOPC to form the HII phase, but DMPC remained lamellar. Measurements of surface tension showed that GrA in vesicles produced a general increase in the rate of adsorption for both phospholipids. When restricted to the interface, however, in preexisting films, GrA with DOPC, but not with DMPC, replicated the ability of the surfactant proteins to promote adsorption of vesicles containing only the lipids. The correlation between the structural and functional effects of GrA with the two phospholipids, and the similar effects on adsorption of GrA with DOPC and the hydrophobic surfactant proteins with the surfactant lipids fit with the model in which SP-B and SP-C facilitate adsorption by stabilizing a rate-limiting intermediate with negative curvature. PMID:16242116

SwissProt search result: AK110012 [KOME

Lifescience Database Archive (English)

Full Text Available ulfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 1e-14 ...

SwissProt search result: AK065733 [KOME

Lifescience Database Archive (English)

Full Text Available lfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 4e-18 ...

SwissProt search result: AK242597 [KOME

Lifescience Database Archive (English)

Full Text Available lfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 5e-19 ...

SwissProt search result: AK103514 [KOME

Lifescience Database Archive (English)

Full Text Available lfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 1e-33 ...

SwissProt search result: AK120333 [KOME

Lifescience Database Archive (English)

Full Text Available lfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 2e-18 ...

SwissProt search result: AK064293 [KOME

Lifescience Database Archive (English)

Full Text Available ulfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 2e-15 ...

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

Science.gov (United States)

Yano, Kanako; Yanagisawa, Takahiro; Mukae, Kyosuke; Niwa, Yasuo; Inoue, Yuko; Moriyasu, Yuji

2015-01-01

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H(+)-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Science.gov (United States)

Yano, Kanako; Yanagisawa, Takahiro; Mukae, Kyosuke; Niwa, Yasuo; Inoue, Yuko; Moriyasu, Yuji

2015-01-01

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H+-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation. PMID:26368310

Palladium nanoparticles anchored on graphene nanosheets: Methanol, ethanol oxidation reactions and their kinetic studies

KAUST Repository

Nagaraju, Doddahalli H.; Devaraj, Sappani; Balaya, Palani

2014-01-01

nanosheets. The electro-catalytic activity of Gra/Pd nanocomposites toward methanol and ethanol oxidation in alkaline medium was evaluated by cyclic voltammetric studies. 1:1 Gra/Pd nanocomposite exhibited good electro-catalytic activity and efficient

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.

Definition of APC presentation of phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate to Vgamma2Vdelta 2 TCR.

Science.gov (United States)

Wei, Huiyong; Huang, Dan; Lai, Xiaomin; Chen, Meiling; Zhong, Weihua; Wang, Richard; Chen, Zheng W

2008-10-01

Although microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) can activate primate Vgamma2Vdelta2 T cells, molecular mechanisms by which HMBPP interacts with Vgamma2Vdelta2 T cells remain poorly characterized. Here, we developed soluble, tetrameric Vgamma2Vdelta2 TCR of rhesus macaques to define HMBPP/APC interaction with Vgamma2Vdelta2 TCR. While exogenous HMBPP was associated with APC membrane in an appreciable affinity, the membrane-associated HMBPP readily bound to the Vgamma2Vdelta2 TCR tetramer. The Vgamma2Vdelta2 TCR tetramer was shown to bind stably to HMBPP presented on membrane by various APC cell lines from humans and nonhuman primates but not those from mouse, rat, or pig. The Vgamma2Vdelta2 TCR tetramer also bound to the membrane-associated HMBPP on primary monocytes, B cells and T cells. Consistently, endogenous phosphoantigen produced in Mycobacterium-infected dendritic cells was transported and presented on membrane, and bound stably to the Vgamma2Vdelta2 TCR tetramer. The capability of APC to present HMBPP for recognition by Vgamma2Vdelta2 TCR was diminished after protease treatment of APC. Thus, our studies elucidated an affinity HMBPP-APC association conferring stable binding to the Vgamma2Vdelta2 TCR tetramer and the protease-sensitive nature of phosphoantigen presentation. The findings defined APC presentation of phosphoantigen HMBPP to Vgamma2Vdelta2 TCR.

The grey relational approach for evaluating measurement uncertainty with poor information

International Nuclear Information System (INIS)

Luo, Zai; Wang, Yanqing; Zhou, Weihu; Wang, Zhongyu

2015-01-01

The Guide to the Expression of Uncertainty in Measurement (GUM) is the master document for measurement uncertainty evaluation. However, the GUM may encounter problems and does not work well when the measurement data have poor information. In most cases, poor information means a small data sample and an unknown probability distribution. In these cases, the evaluation of measurement uncertainty has become a bottleneck in practical measurement. To solve this problem, a novel method called the grey relational approach (GRA), different from the statistical theory, is proposed in this paper. The GRA does not require a large sample size or probability distribution information of the measurement data. Mathematically, the GRA can be divided into three parts. Firstly, according to grey relational analysis, the grey relational coefficients between the ideal and the practical measurement output series are obtained. Secondly, the weighted coefficients and the measurement expectation function will be acquired based on the grey relational coefficients. Finally, the measurement uncertainty is evaluated based on grey modeling. In order to validate the performance of this method, simulation experiments were performed and the evaluation results show that the GRA can keep the average error around 5%. Besides, the GRA was also compared with the grey method, the Bessel method, and the Monte Carlo method by a real stress measurement. Both the simulation experiments and real measurement show that the GRA is appropriate and effective to evaluate the measurement uncertainty with poor information. (paper)

Coloración de gráficas suaves

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Pedro Lara Velázquez

2015-08-01

Full Text Available En este trabajo se propone un modelo de Coloración en Gráficas Suaves donde se colorea con base en ponderaciones sobre las aristas de la gráfica. Se muestra que este modelo es muy flexible e incluye otros problemas similares, tales como los problemas de Coloración Mínima, Coloración Equitativa, Coloración de Gráficas Débiles y Coloración Robusta. Se proponen también un modelo binario lineal de solución y algunas instancias de prueba.

Enhanced salt resistance in apple plants overexpressing a Malus vacuolar Na+/H+ antiporter gene is associated with differences in stomatal behavior and photosynthesis.

Science.gov (United States)

Li, Chao; Wei, Zhiwei; Liang, Dong; Zhou, Shasha; Li, Yonghong; Liu, Changhai; Ma, Fengwang

2013-09-01

High salinity is a major abiotic factor that limits crop production. The dwarfing apple rootstock M.26 is sensitive to such stress. To obtain an apple that is adaptable to saline soils, we transformed this rootstock with a vacuolar Na(+)/H(+) antiporter, MdNHX1. Differences in salt tolerance between transgenic and wild-type (WT) rootstocks were examined under field conditions. We also compared differences when 'Naganofuji No. 2' apple was grafted onto these transgenic or WT rootstocks. Plants on the transgenic rootstocks grew well during 60 d of mild stress (100 mM NaCl) while the WT exhibited chlorosis, inhibited growth and even death. Compared with the untreated control, the stomatal density was greater in both non-grafted and grafted WT plants exposed to 200 mM NaCl. In contrast, that density was significantly decreased in leaves from grafted transgenic plants. At 200 mM NaCl, net photosynthesis, stomatal conductance, intercellular CO2 concentration, and chlorophyll contents were markedly reduced in the WT, whereas the declines in those values were only minor in similarly stressed transgenic plants. Therefore, we conclude that overexpressing plants utilize a better protective mechanism for retaining higher photosynthetic capacity. Furthermore, this contrast in tolerance and adaptability to stress is linked to differences in stomatal behavior and photosynthetic rates. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

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.

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.

Decreased proteinase A excretion by strengthening its vacuolar sorting and weakening its constitutive secretion in Saccharomyces cerevisiae.

Science.gov (United States)

Chen, Yefu; Song, Lulu; Han, Yueran; Liu, Mingming; Gong, Rui; Luo, Weiwei; Guo, Xuewu; Xiao, Dongguang

2017-01-01

Proteinase A (PrA), encoded by PEP4 gene, is detrimental to beer foam stability. There are two transport pathways for the new synthesized PrA in yeast, sorting to the vacuole normally, or excreting out of the cells under stress conditions. They were designated as the Golgi-to-vacuole pathway and the constitutive secretory pathway, respectively. To reduce PrA excretion in some new way instead of its coding gene deletion, which had a negative effect on cell metabolism and beer fermentation, we modified the PrA transport based on these above two pathways. In the Golgi-to-vacuole pathway, after the verification that Vps10p is the dominant sorting receptor for PrA Golgi-to-vacuolar transportation by VPS10 deletion, VPS10 was then overexpressed. Furthermore, SEC5, encoding exocyst complexes' central subunit (Sec5p) in the constitutive secretory pathway, was deleted. The results show that PrA activity in the broth fermented with WGV10 (VPS10 overexpressing strain) and W∆SEC5 (SEC5 deletion strain) was lowered by 76.96 and 32.39%, compared with the parental strain W303-1A, at the end of main fermentation. There are negligible changes in fermentation performance between W∆SEC5 and W303-1A, whereas, surprisingly, WGV10 had a significantly improved fermentation performance compared with W303-1A. WGV10 has an increased growth rate, resulting in higher biomass and faster fermentation speed; finally, wort fermentation is performed thoroughly. The results show that the biomass production of WGV10 is always higher than that of W∆SEC5 and W303-1A at all stages of fermentation, and that ethanol production of WGV10 is 1.41-fold higher than that of W303-1A. Obviously, VPS10 overexpression is beneficial for yeast and is a more promising method for reduction of PrA excretion.

Phytoremediation potential of Arabidopsis thaliana, expressing ectopically a vacuolar proton pump, for the industrial waste phosphogypsum.

Science.gov (United States)

Khoudi, Habib; Maatar, Yafa; Brini, Faïçal; Fourati, Amine; Ammar, Najoua; Masmoudi, Khaled

2013-01-01

transgenic A. thaliana lines were more Cd-tolerant than the WT plants. These results suggested that ectopic expression of a vacuolar proton pump in A. thaliana plants can lead to various biotechnological applications including the phytoremediation of industrial wastes.

Using an Evaluability Assessment To Select Methods for Evaluating State Technology Development Programs: The Case of the Georgia Research Alliance.

Science.gov (United States)

Youtie, Jan; Bozeman, Barry; Shapira, Philip

1999-01-01

Describes an evaluability assessment of the Georgia Research Alliance (GRA), a technology development program. Presents the steps involved in conducting an evaluability assessment, including development of an understanding of the program and its stakeholders. Analyzes and compares different methods by which the GRA could be evaluated. (SLD)

Preliminary Iron Distribution on Vesta

Science.gov (United States)

Mittlefehldt, David W.; Mittlefehldt, David W.

2013-01-01

The distribution of iron on the surface of the asteroid Vesta was investigated using Dawn's Gamma Ray and Neutron Detector (GRaND) [1,2]. Iron varies predictably with rock type for the howardite, eucrite, and diogenite (HED) meteorites, thought to be representative of Vesta. The abundance of Fe in howardites ranges from about 12 to 15 wt.%. Basaltic eucrites have the highest abundance, whereas, lower crustal and upper mantle materials (cumulate eucrites and diogenites) have the lowest, and howardites are intermediate [3]. We have completed a mapping study of 7.6 MeV gamma rays produced by neutron capture by Fe as measured by the bismuth germanate (BGO) detector of GRaND [1]. The procedures to determine Fe counting rates are presented in detail here, along with a preliminary distribution map, constituting the necessary initial step to quantification of Fe abundances. We find that the global distribution of Fe counting rates is generally consistent with independent mineralogical and compositional inferences obtained by other instruments on Dawn such as measurements of pyroxene absorption bands by the Visual and Infrared Spectrometer (VIR) [4] and Framing Camera (FC) [5] and neutron absorption measurements by GRaND [6].

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV-3) and possible latency of CyHV-3 by temperature shift in the cells.

Science.gov (United States)

Imajoh, M; Fujioka, H; Furusawa, K; Tamura, K; Yamasaki, K; Kurihara, S; Yamane, J; Kawai, K; Oshima, S

2015-06-01

A new cell line named CCF-K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV-3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV-3-infected CCF-K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV-3 was produced stably in CCF-K104 cells over 30 viral passages. Our findings showed that CCF-K104 is a useful cell line for isolation and productive replication of CyHV-3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real-time PCR showed that CyHV-3 was present with low viral DNA loads, suggesting that CyHV-3 may establish latent infection in CCF-K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV-3 genome arranged in a head-to-tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV-3. © 2014 John Wiley & Sons Ltd.

A grey relational analytical approach to orange peel filler particulates for tapped density experiments of green composite reinforcements

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Oluwaseyi Ayodele Ajibade

2016-08-01

Full Text Available Monitoring density changes during transportation of composite fillers is the principal reason for composite industries implementing the tapped density concept. Till date, very sparse literature information exists on tapped density optimisation for orange peel particles (OPPs. A unique application of grey relational analysis (GRA in the optimisation of tapped process parameters for OPPs is contributed in this paper. Experimental results on the principal process parameters indicate G1H1I2J3 as the best experimental run, which translates to 257.956 g and 78.076 cm3 as well as 254.939 g and 72.94 cm3 for masses and volumes of the 0.425 and 0.600 mm OPPs, respectively. In addition, Taguchi method was applied to arrive at an optimal parametric setting of G2H2I1J1 for comparative purposes which translate to 257.723 g and 75.031 cm3 for mass and volume of the 0.425 OPPs, as well as 254.952 g and 77.982 cm3 for the mass and volume of 0.600 mm OPPs. By comparison, the GRA values produced positive percentage improvement over other optimal values. The unique contribution of this paper are principally the (i application of GRA in a novel manner, incorporating harmonic mean in factor-level determination and computation of S/N responses; (ii development of new indices of tapped density; and (iii introduction of economic factors in tapped density computations, incorporating inflation and interest factors. The practical utility of the demonstrated approach lies in reducing the uncertainties about density measurements in the transportation of green fillers for use as composite reinforcements.

Differential expression in Phanerochaete chrysosporium of membrane- associated proteins relevant to lignin degradation

Science.gov (United States)

Semarjit Shary; Alexander N. Kapich; Ellen A. Panisko; Jon K. Magnuson; Daniel Cullen; Kenneth E. Hammel

2008-01-01

Fungal lignin-degrading systems likely include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, production of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Little is known about the nature or regulation of these membrane-associated components. We grew...

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

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

2015-12-01

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

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.

Generation Risk Assessment Using Fault Trees and Turbine Cycle Simulation: Case Studies

International Nuclear Information System (INIS)

Heo, Gyun Young; Park, Jin Kyun

2009-01-01

Since 2007, Korea Atomic Energy Research Institute and Kyung Hee University have collaborated on the development of the framework to quantify human errors broken out during the test and maintenance (T and M) in secondary systems of nuclear power plants (NPPs). The project entitled 'Development of Causality Analyzer for Maintenance/Test Tasks in Nuclear Power Plants' for OPR1000 on the basis of the proposed framework is still on-going, and will come to fruition by 2010. The overall concept of GRA-HRE (Generation Risk Assessment for Human Related Events) which is the designation of the framework, and the quantification methods for evaluating risk and electric loss have introduced in other references. The originality emerged while implementing GRA-HRE could be evaluated in view of (1) recognizing the relative importance of human errors comparing with other types of mechanical and/or electrical failures, (2) providing the top-down path of the propagation of human errors by designating top events in the fault tree model as trip signals, and (3) analyzing electric loss using turbine cycle simulation. Recently, we were successfully to illustrate the applicability of GRA-HRE by simulating several abnormalities. Since the detailed methodologies were released enough to follow up, this paper is going to only exemplify the case studies

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole*

Science.gov (United States)

Ho, Cheuk Y.; Choy, Christopher H.; Wattson, Christina A.; Johnson, Danielle E.; Botelho, Roberto J.

2015-01-01

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. PMID:25713145

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

Science.gov (United States)

Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

2016-02-03

Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.

New rurality, traditional music and tourist experience

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Margarida Vaz

2017-03-01

Full Text Available Fernando Lopes-Graça, a prolific Portuguese composer, joined the French musicologist Michel Giacometti (1929-1990 in the effort of collecting and cataloguing popular songs, culminating in a collection of recordings, unique in Europe, which covered all Portugal. In the context of this paper we intend to show how the work of Lopes-Graça, challenging the traditional music, can allow a touring through traditions, while allowing design a cultural environment for the most demanding tourists. The purpose of this research is to develop a tourism product in the near future based on Lopes-Graça and Giacometti’s (musical and spatial journey through Portuguese traditional music, which portray a rural Portugal in all its dimensions including beliefs, uses and activities.

Kartografski prikazi sakralnih objekata i groblja na katastarskim planovima stare izmjere Bosne i Hercegovine

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Nedim Tuno

2015-01-01

Full Text Available Rad se bavi kartografskim prikazima objekta vjerskog karaktera i groblja u Bosni i Hercegovini, na katastarskim planovima izrađenim tijekom uprave Austro-Ugarske. Pokazano je kako se kartografsko prikazivanje na tim planovima mijenjalo na temelju zbirki kartografskih znakova koje su objavljene potkraj 19. i početkom 20. stoljeća. Proučavanjem prikupljene izvorne arhivske građe identificirani su relevantni kartografski izvori, izrađeni u različitim mjerilima i različitim metodama, na kojima su kartografirane vjerske građevine. Na temelju provedenih istraživanja, autori su pomoću relevantne kartografske građe sagledali najvažnije aspekte kartografskih prikazivanja sakralnih objekata različitih religijskih zajednica u Bosni i Hercegovini.

An Empirical Analysis of the Navy Junior Reserve Officer Training Corps (NJROTC)

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1987-12-01

o_ _ion For NTIS GRA&I DTIC TAB 0 Ula•ncnmwood [] , ,, Juutiriou~iozl Dis !tribution/ Availability (,’ odk 3 Alra .taI / Diat ~~za V. TABLE OF CONTENTS...followingq: I. The overall total 2. The row totals 3. The column totals "Supertables" were also extensively used. A supertable is essentially a collection

Epizootic vacuolar myelinopathy of the central nervous system of bald eagles (Haliaeetus leucocephalus) and American coots (Fulica americana)

Science.gov (United States)

Thomas, N.J.; Meteyer, C.U.; Sileo, L.

1998-01-01

toxicity from hexachlorophene, triethyltin, bromethalin, isonicotinic acid hydrazide, and certain exotic plant toxins; however, despite exhaustive testing, no etiology was determined for the DeGray Lake mortality events. This is the first report of vacuolar myelinopathy associated with spontaneous mortality in wild birds.

La lectura en el aula. Qué se hace, qué se debe hacer y qué se puede hacer

OpenAIRE

Pablo Lorente Muñoz

2011-01-01

Emilio Sánchez Miguel (Coord.). La lectura en el aula. Qué se hace, qué se debe hacer y qué se puede hacer. Graó, col. Crítica y Fundamentos, 27. Barcelona, 2010. 382 págs. ISBN: 978-84-7827-892-3

Palladium nanoparticles anchored on graphene nanosheets: Methanol, ethanol oxidation reactions and their kinetic studies

KAUST Repository

Nagaraju, Doddahalli H.

2014-12-01

Palladium nanoparticles decorated graphene (Gra/Pd nanocomposite) was synthesized by simultaneous chemical reduction of graphene oxide and palladium salt in a single step. The negatively charged graphene oxide (GO) facilitates uniform distribution of Pd2+ ions onto its surface. The subsequent reduction by hydrazine hydrate provides well dispersed Pd nanoparticles decorated graphene. Different amount of Pd nanoparticles on graphene was synthesized by changing the volume to weight ratio of GO to PdCl2. X-ray diffraction studies showed FCC lattice of Pd with predominant (1 1 1) plane. SEM and TEM studies revealed that thin graphene nanosheets are decorated by Pd nanoparticles. Raman spectroscopic studies revealed the presence of graphene nanosheets. The electro-catalytic activity of Gra/Pd nanocomposites toward methanol and ethanol oxidation in alkaline medium was evaluated by cyclic voltammetric studies. 1:1 Gra/Pd nanocomposite exhibited good electro-catalytic activity and efficient electron transfer. The kinetics of electron transfer was studied using chronoamperometry. Improved electro-catalytic activity of 1:1 Gra/Pd nanocomposite toward alcohol oxidation makes it as a potential anode for the alcohol fuel cells. © 2014 Elsevier Ltd.

A Neospora caninum vaccine using recombinant proteins fails to prevent foetal infection in pregnant cattle after experimental intravenous challenge.

Science.gov (United States)

Hecker, Yanina P; Cóceres, Verónica; Wilkowsky, Silvina E; Jaramillo Ortiz, José M; Morrell, Eleonora L; Verna, Andrea E; Ganuza, Agustina; Cano, Dora B; Lischinsky, Lilian; Angel, Sergio O; Zamorano, Patricia; Odeón, Anselmo C; Leunda, María R; Campero, Carlos M; Morein, Bror; Moore, Dadín P

2014-12-15

The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20 and rNcGRA7 recombinant proteins formulated with immune stimulating complexes (ISCOMs) in pregnant heifers against vertical transmission of Neospora caninum. Twelve pregnant heifers were divided into 3 groups of 4 heifers each, receiving different formulations before mating. Immunogens were administered twice subcutaneously: group A animals were inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B animals received ISCOM-MATRIX (without antigen) and group C received sterile phosphate-buffered saline (PBS) only. The recombinant proteins were expressed in Escherichia coli and purified nickel resin. All groups were intravenously challenged with the NC-1 strain of N. caninum at Day 70 of gestation and dams slaughtered at week 17 of the experiment. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 prior to the challenge. Following immunization, an statistically significant increase of antibodies against rNcSAG1 and rNcHSP20 in all animals of group A was detected compared to animals in groups B and C at weeks 5, 13 and 16 (P0.001). There were no differences in IFN-γ production among the experimental groups at any time point (P>0.05). Transplacental transmission was determined in all foetuses of groups A, B and C by Western blot, immunohistochemistry and nested PCR. This work showed that rNcSAG1, rNcHSP20 and rNcGRA7 proteins while immunogenic in cattle failed to prevent the foetal infection in pregnant cattle challenged at Day 70 of gestation. Copyright © 2014 Elsevier B.V. All rights reserved.

Ab initio study of the binding of collagen amino acids to graphene and A-doped (A = H, Ca) graphene

Energy Technology Data Exchange (ETDEWEB)

Cazorla, Claudio, E-mail: c.silva@ucl.ac.u

2010-09-30

We present a theoretical study of the binding of collagen amino acids (AA, namely glycine, Gly; proline, Pro; and hydroxyproline, Hyp) to graphene (Gr), Ca-doped graphene and graphane (Gra) using density functional theory calculations and ab initio molecular dynamics (AIMD) simulations. It is found that binding of Gly, Pro and Hyp to Gr and Gra is thermodynamically favorable yet dependent on the amino acid orientation and always very weak (adsorption energies E{sub ads} range from -90 to -20 meV). AIMD simulations reveal that room-temperature thermal excitations are enough to induce detachment of Gly and Pro from Gr and of all three amino acids from Gra. Interestingly, we show that collagen AA binding to Gr is enhanced dramatically by doping the carbon surface with calcium atoms (corresponding E{sub ads} values decrease by practically two orders of magnitude with respect to the non-doped case). This effect is result of electronic charge transfers from the Ca impurity (donor) to Gr (acceptor) and the carboxyl group (COOH) of the amino acid (acceptor). The possibility of using Gr and Gra as nanoframes for sensing of collagen amino acids has also been investigated by performing electronic density of states analysis. It is found that, whether Gr is hardly sensitive, the electronic band gap of Gra can be modulated by attaching different number and species of AAs onto it. The results presented in this work provide fundamental insights on the quantum interactions of collagen protein components with carbon-based nanostructures and can be useful for developments in bio and nanotechnology fields.

Obesity Exposure Across the Lifespan on Ovarian Cancer Pathogenesis

Science.gov (United States)

2014-06-01

channel, KQT-like subfamily, member 2 -3.1607 0.00179 0.078683 FLJ20184 hypothetical protein FLJ20184 -3.15415 0.001829 0.079906 USH1C Usher syndrome ...ERGIC2 ERGIC and golgi 2 2.981139 0.003188 0.097932 BBS4 Bardet-Biedl syndrome 4 2.981241 0.003187 0.097932 MARF5 membrane-associated ring

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

Science.gov (United States)

Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

2015-04-10

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparison of canny and V1 neural network based edge detectors applied to road extraction

CSIR Research Space (South Africa)

Hauptfleisch, AC

2006-11-01

Full Text Available and vertically, for succes- sive pixels p and q that satisfy the distance and gradient orien- tation conditions. The conditions can be summarised as follows (See Figure 3): • The gradient at p must be approximately equal to the gra- dient at q plus 180...

Malaria Parasite CLAG3, a Protein Linked to Nutrient Channels, Participates in High Molecular Weight Membrane-Associated Complexes in the Infected Erythrocyte.

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Kayvan Zainabadi

Full Text Available Malaria infected erythrocytes show increased permeability to a number of solutes important for parasite growth as mediated by the Plasmodial Surface Anion Channel (PSAC. The P. falciparum clag3 genes have recently been identified as key determinants of PSAC, though exactly how they contribute to channel function and whether additional host/parasite proteins are required remain unknown. To begin to answer these questions, I have taken a biochemical approach. Here I have used an epitope-tagged CLAG3 parasite to perform co-immunoprecipitation experiments using membrane fractions of infected erythrocytes. Native PAGE and mass spectrometry studies reveal that CLAG3 participate in at least three different high molecular weight complexes: a ~720kDa complex consisting of CLAG3, RHOPH2 and RHOPH3; a ~620kDa complex consisting of CLAG3 and RHOPH2; and a ~480kDa complex composed solely of CLAG3. Importantly, these complexes can be found throughout the parasite lifecycle but are absent in untransfected controls. Extracellular biotin labeling and protease susceptibility studies localize the 480kDa complex to the erythrocyte membrane. This complex, likely composed of a homo-oligomer of 160kDa CLAG3, may represent a functional subunit, possibly the pore, of PSAC.

Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H(+)-pyrophosphatase gene (SbVPPase) from Sorghum bicolor.

Science.gov (United States)

Anjaneyulu, Ediga; Reddy, Palle Surender; Sunita, Merla Srilakshmi; Kishor, Polavarapu B Kavi; Meriga, Balaji

2014-06-15

A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80-90% homology at the nucleotide and 85-95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50-70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25-100% in transgenics, while malondialdehyde (MDA) showed a 2-4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5-2.5-fold higher Na(+) and 0.4-0.8-fold higher K(+) levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress. Copyright © 2014 Elsevier GmbH. All rights reserved.

31P-Nuclear Magnetic Resonance Determination of Phosphate Compartmentation in Leaves of Reproductive Soybeans (Glycine max L.) as Affected by Phosphate Nutrition 1

Science.gov (United States)

Lauer, Michael J.; Blevins, Dale G.; Sierzputowska-Gracz, Hanna

1989-01-01

Most leaf phosphorus is remobilized to the seed during reproductive development in soybean. We determined, using 31P-NMR, the effect phosphorus remobilization has on vacuolar inorganic phosphate pool size in soybean (Glycine max [L.] Merr.) leaves with respect to phosphorus nutrition and plant development. Phosphate compartmentation between cytoplasmic and vacuolar pools was observed and followed in intact tissue grown hydroponically, at the R2, R4, and R6 growth stages. As phosphorus in the nutrient solution decreased from 0.45 to 0.05 millimolar, the vacuolar phosphate peak became less prominent relative to cytoplasmic phosphate and hexose monophosphate peaks. At a nutrient phosphate concentration of 0.05 millimolar, the vacuolar phosphate peak was not detectable. At higher levels of nutrient phosphate, as plants progressed from the R2 to the R6 growth stage, the vacuolar phosphate peak was the first to disappear, suggesting that storage phosphate was remobilized to a greater extent than metabolic phosphate. Under suboptimal phosphate nutrition (≤ 0.20 millimolar), the hexose monophosphate and cytoplasmic phosphate peaks declined earlier in reproductive development than when phosphate was present in optimal amounts. Under low phosphate concentrations (0.05 millimolar) cytoplasmic phosphate was greatly reduced. Carbon metabolism was coincidently disrupted under low phosphate nutrition as shown by the appearance of large, prominent starch grains in the leaves. Cytoplasmic phosphate, and leaf carbon metabolism dependent on it, are buffered by vacuolar phosphate until late stages of reproductive growth. Images Figure 4 PMID:16666705

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

Regulation of transport processes across the tonoplast membrane

Directory of Open Access Journals (Sweden)

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.

Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

Science.gov (United States)

Mellouk, Nora; Enninga, Jost

2016-01-01

Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

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.

Morphological classification of plant cell deaths

DEFF Research Database (Denmark)

van Doorn, W.G.; Beers, E.P.; Dangl, J.L.

2011-01-01

, 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....... 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...

Proceedings of the Natick Science Symposium (3rd) Held in Natick, Massachusetts on 5-6 June 1990

Science.gov (United States)

1990-08-01

ring, in egg cocccn st.."Llct:’..rres, as adhesives, ar.d L""l prey capture. ’There has been scme data collected on the physical prq:::~ies of...dimi.ni.shin;J effects, such as nonenzynatic browning. For example, sugar, milk ani eggs typically added to ~ial breads to enhance acceptability are not...Chicken Stew Gravy Pork Sausage Pa~ Pork WjRice ~ Meatballs /~ Meat Balls/~ Beef I Diced W/GraV’/ Co:rned Beef Hash Chicken loaf Chicken arrl Rice

Speech Processing in Realistic Battlefield Environments (Le Traitement de la Parole en Environnement de Combat Realiste)

Science.gov (United States)

2009-04-01

accessed utilizing 4 Watkins Johnson (WJ-8611) receivers connected to a high quality 24 channel Mark Of The Unicorn 24I/O analog-to-digital... Government Reports Announcements & Index (GRA&I) STAR peut être consulté en ligne au localisateur de ressources publié par le National Technical...given in the following journals: Scientific and Technical Aerospace Reports (STAR) Government Reports Announcements & Index (GRA&I) STAR is available

Antarctic Meteorite Newsletter

Science.gov (United States)

Lindstrom, Marilyn

2000-01-01

This newsletter contains something for everyone! It lists classifications of about 440 meteorites mostly from the 1997 and 1998 ANSMET (Antarctic Search for Meteorites) seasons. It also gives descriptions of about 45 meteorites of special petrologic type. These include 1 iron, 17 chondrites (7 CC, 1 EC, 9 OC) and 27 achondrites (25 HED, UR). Most notable are an acapoloite (GRA98028) and an olivine diogenite (GRA98108).

Conceptual design study of 1985 commercial tilt rotor transports. Volume 3. STOL design summary. Final report

Energy Technology Data Exchange (ETDEWEB)

Sambell, K.W.

1976-04-01

A conceptual design study is presented of 1,985 commercial tilt rotor STOL transports for a NASA 200 n. mi. (370 km) STOL Mission. A 100-passenger STOL Variant (Bell D313) of the Phase I VTOL Tilt Rotor Aircraft is defined. Aircraft characteristics are given; with the aircraft redesigned to meet 2,000-foot (610 m) field criteria, with emphasis on low fuel consumption and low direct operating cost. The 100-passenger STOL Tilt Rotor Aircraft was analyzed for performance, weights, economics, handling qualities, noise footprint and aeroelastic stability. (GRA)

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

Science.gov (United States)

Millet, Arnaud; Martin, Katherine R.; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A.; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

2015-01-01

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

Multi-response optimization of process parameters in friction stir welded AM20 magnesium alloy by Taguchi grey relational analysis

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Prakash Kumar Sahu

2015-03-01

Full Text Available The purpose of this paper is to optimize the process parameter to get the better mechanical properties of friction stir welded AM20 magnesium alloy using Taguchi Grey relational analysis (GRA. The considered process parameters are welding speed, tool rotation speed, shoulder diameter and plunging depth. The experiments were carried out by using Taguchi's L18 factorial design of experiment. The processes parameters were optimized and ranked the parameters based on the GRA. The percentage influence of each process parameter on the weld quality was also quantified. A validation experimental run was conducted using optimal process condition, which was obtained from the analysis, to show the improvement in mechanical properties of the joint. This study also shows the feasibility of the GRA with Taguchi technique for improvement in welding quality of magnesium alloy.

Radiotelemetry Tracking at Lake Conway, Florida.

Science.gov (United States)

1982-08-01

objec- tives of this project could not have been successfully met. Accession For NTIS GRA&I DTIC TAB Unannounced F1 Justification By Distribution...Use in Studies of Movements of Fishes," Trans-actions of the American Fisheries Society, Vol 95, No. 4, pp 350-356. A3 Holand , B. 1974. "Ultrasonic

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

Directory of Open Access Journals (Sweden)

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

Semaphorin 3 C drives epithelial-to-mesenchymal transition, invasiveness, and stem-like characteristics in prostate cells.

Science.gov (United States)

Tam, Kevin J; Hui, Daniel H F; Lee, Wilson W; Dong, Mingshu; Tombe, Tabitha; Jiao, Ivy Z F; Khosravi, Shahram; Takeuchi, Ario; Peacock, James W; Ivanova, Larissa; Moskalev, Igor; Gleave, Martin E; Buttyan, Ralph; Cox, Michael E; Ong, Christopher J

2017-09-13

Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition (EMT) and stem-like phenotypes in prostate cells. In the present study we show that ectopic expression of SEMA3C in RWPE-1 promotes the upregulation of EMT and stem markers, heightened sphere-formation, and cell plasticity. In addition, we show that SEMA3C promotes migration and invasion in vitro and cell dissemination in vivo.

Endolysosomal pathway activity protects cells from neurotoxic TDP-43

Directory of Open Access Journals (Sweden)

Christine Leibiger

2018-03-01

Full Text Available The accumulation of protein aggregates in neurons is a typical pathological hallmark of the motor neuron disease amyotrophic lateral sclerosis (ALS and of frontotemporal dementia (FTD. In many cases, these aggregates are composed of the 43 kDa TAR DNA-binding protein (TDP‑43. Using a yeast model for TDP‑43 proteinopathies, we observed that the vacuole (the yeast equivalent of lysosomes markedly contributed to the degradation of TDP‑43. This clearance occurred via TDP‑43-containing vesicles fusing with the vacuole through the concerted action of the endosomal-vacuolar (or endolysosomal pathway and autophagy. In line with its dominant role in the clearance of TDP‑43, endosomal-vacuolar pathway activity protected cells from the detrimental effects of TDP‑43. In contrast, enhanced autophagy contributed to TDP‑43 cytotoxicity, despite being involved in TDP‑43 degradation. TDP‑43’s interference with endosomal-vacuolar pathway activity may have two deleterious consequences. First, it interferes with its own degradation via this pathway, resulting in TDP‑43 accumulation. Second, it affects vacuolar proteolytic activity, which requires endosomal-vacuolar trafficking. We speculate that the latter contributes to aberrant autophagy. In sum, we propose that ameliorating endolysosomal pathway activity enhances cell survival in TDP‑43-associated diseases.

Cytosolic access of intracellular bacterial pathogens: the Shigella paradigm

Directory of Open Access Journals (Sweden)

Nora eMellouk

2016-04-01

Full Text Available Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

GRAÚNA: flights and sings of children “Quilombola’s” Curriculum

Directory of Open Access Journals (Sweden)

Marisa Terezinha Rosa Valladares

2016-06-01

Full Text Available Problematize (impossibilities of flights and chants in a curriculum lived by children in the areas of a quilombola community, in their first school years, motivated this work. The methodological composition, in a concept of daily research, encompassed conversations and narrations in recesses, classes and visits to the Graúna School, Itapemirim – Espírito Santo. Understanding that a lived curriculum adds teaching plans and learning, experienced in the daily academic life, invading community life, to beyond the school boundaries, structuring negotiations in conversations, authors whose comprehensions support these questions are invited to formulation of concepts, such as community/school. Graúna’s community holds peculiarity of quilombola registration by ancestry of its people, without a resistance movement for territory maintenance and without quilombola identity admitted by cultural tradition. The school’s curricular act, in pedagogical gesture, elaborates the entanglement community/school to (recreate the quilombola identity in the community. The reality of the Graúna children moves through figurative and concrete sights, where the open gates of the school do not encage hope, but give freedom for the little wings to prepare to fly over the world, chanting identities and well-being of stories lived in different times, in the impetus provided by a community/school, called Graúna – not by chance. Problematizar (impossibilidades de voos e (encantos num currículo vivido por crianças em geografias de uma comunidade quilombola, nos anos iniciais escolares, motivou este trabalho. A composição metodológica, numa concepção de pesquisa com cotidiano, abarcou conversações e narrativas em recreios, aulas e visitas à escola de Graúna, Itapemirim – Espírito Santo. Entendendo que um currículo vivido soma planos de ensino e aprendizagens, experimentadas no cotidiano escolar, invadindo vida de comunidade, para além muros de escola

Compromise of the hypoglycaemic effect of chlorpropamide by the ...

African Journals Online (AJOL)

Severe wide spread of vacuolar degeneration and moderate polymorphonuclear cell degeneration of the liver and vacuolar congestion of the cortex medulla of the kidney was observed with the co-administration. The outcome of this study revealed that coadministration of V. amygdalina and chlorpropamide does not have ...

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

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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.

Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3.

Science.gov (United States)

Nagel, Marie-Kristin; Kalinowska, Kamila; Vogel, Karin; Reynolds, Gregory D; Wu, Zhixiang; Anzenberger, Franziska; Ichikawa, Mie; Tsutsumi, Chie; Sato, Masa H; Kuster, Bernhard; Bednarek, Sebastian Y; Isono, Erika

2017-08-22

Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta , indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the ...

Integration of Tax Administration to Curb Import and Domestic Tax Evasions in Ghana

OpenAIRE

John Adu Kwame; Eric Tutu Tchao; Kwasi Poku

2013-01-01

As part of the Government of Ghana’s plans to maximize tax mobilization, it recently integrated its Regional Collection Agencies (RCA) namely; the Internal Revenue Service (IRS), Customs Excise and Preventive service (CEPS) and the Value Added Tax (VAT) Services into the Ghana Revenue Authority (GRA). This research aims to find out whether Ghana’s tax administration reform of integrating the RCA into GRA has dealt with the inefficiencies in tax administration with respect to personal income t...

Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field

KAUST Repository

Schilling, Rhiannon K.

2013-11-22

Cereal varieties with improved salinity tolerance are needed to achieve profitable grain yields in saline soils. The expression of AVP1, an Arabidopsis gene encoding a vacuolar proton pumping pyrophosphatase (H+-PPase), has been shown to improve the salinity tolerance of transgenic plants in greenhouse conditions. However, the potential for this gene to improve the grain yield of cereal crops in a saline field has yet to be evaluated. Recent advances in high-throughput nondestructive phenotyping technologies also offer an opportunity to quantitatively evaluate the growth of transgenic plants under abiotic stress through time. In this study, the growth of transgenic barley expressing AVP1 was evaluated under saline conditions in a pot experiment using nondestructive plant imaging and in a saline field trial. Greenhouse-grown transgenic barley expressing AVP1 produced a larger shoot biomass compared to segregants, as determined by an increase in projected shoot area, when grown in soil with 150 mm NaCl. This increase in shoot biomass of transgenic AVP1 barley occurred from an early growth stage and also in nonsaline conditions. In a saline field, the transgenic barley expressing AVP1 also showed an increase in shoot biomass and, importantly, produced a greater grain yield per plant compared to wild-type plants. Interestingly, the expression of AVP1 did not alter barley leaf sodium concentrations in either greenhouse- or field-grown plants. This study validates our greenhouse-based experiments and indicates that transgenic barley expressing AVP1 is a promising option for increasing cereal crop productivity in saline fields. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field

KAUST Repository

Schilling, Rhiannon K.; Marschner, Petra; Shavrukov, Yuri N.; Berger, Bettina; Tester, Mark A.; Roy, Stuart John; Plett, Darren Craig

2013-01-01

Cereal varieties with improved salinity tolerance are needed to achieve profitable grain yields in saline soils. The expression of AVP1, an Arabidopsis gene encoding a vacuolar proton pumping pyrophosphatase (H+-PPase), has been shown to improve the salinity tolerance of transgenic plants in greenhouse conditions. However, the potential for this gene to improve the grain yield of cereal crops in a saline field has yet to be evaluated. Recent advances in high-throughput nondestructive phenotyping technologies also offer an opportunity to quantitatively evaluate the growth of transgenic plants under abiotic stress through time. In this study, the growth of transgenic barley expressing AVP1 was evaluated under saline conditions in a pot experiment using nondestructive plant imaging and in a saline field trial. Greenhouse-grown transgenic barley expressing AVP1 produced a larger shoot biomass compared to segregants, as determined by an increase in projected shoot area, when grown in soil with 150 mm NaCl. This increase in shoot biomass of transgenic AVP1 barley occurred from an early growth stage and also in nonsaline conditions. In a saline field, the transgenic barley expressing AVP1 also showed an increase in shoot biomass and, importantly, produced a greater grain yield per plant compared to wild-type plants. Interestingly, the expression of AVP1 did not alter barley leaf sodium concentrations in either greenhouse- or field-grown plants. This study validates our greenhouse-based experiments and indicates that transgenic barley expressing AVP1 is a promising option for increasing cereal crop productivity in saline fields. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

A Novel Evaluation Approach for Tourist Choice of Destination Based on Grey Relation Analysis

Directory of Open Access Journals (Sweden)

Xudong Guo

2016-01-01

Full Text Available The decision-making process of choosing an ideal tourism destination is influenced by a number of psychological and nonpsychological variables. Tourists need a method to quickly and easily select a suitable destination. Driven by this practical decision issue, a novel approach of tourist destination evaluation, grey relation analysis (GRA, is developed and applied to the ranking evaluation of Taiwan tourism destinations in China. In the evaluating process, we apply entropy to calculate the weight of each index, which is a more objective method of calculating weights. The results of the study indicate that although the same size is small and the distribution of data is unknown, GRA can still be successfully used in evaluating tourist destinations. In addition, we compare the GRA results with the Technique for Order Preference by Similarity to Ideal Solution (TOPSIS and show that more accurate ranking results can be obtained.

GSK-3α is a central regulator of age-related pathologies in mice.

Science.gov (United States)

Zhou, Jibin; Freeman, Theresa A; Ahmad, Firdos; Shang, Xiying; Mangano, Emily; Gao, Erhe; Farber, John; Wang, Yajing; Ma, Xin-Liang; Woodgett, James; Vagnozzi, Ronald J; Lal, Hind; Force, Thomas

2013-04-01

Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

The Combined Application of Fault Trees and Turbine Cycle Simulation in Generation Risk Assessment

International Nuclear Information System (INIS)

Heo, Gyun Young; Park, Jin Kyun

2009-01-01

The paper describes a few ideas developed for the framework to quantify human errors taking place during the test and maintenance (T and M) in a secondary system of nuclear power plants, which was presented in the previous meeting. GRA-HRE (Generation Risk Assessment for Human Related Events) is composed of four essential components, the human error interpreter, the frequency estimator, the risk estimator, and the derate estimator. The proposed GRA gave emphasis on explicitly considering human errors, performing fault tree analysis including the entire balance-of-plant side, and quantifying electric loss under abnormal plant configurations. In terms of the consideration of human errors, it was hard to distinguish the effects of human errors from other failure modes in the conventional GRA because the human errors were implicitly involved in mechanical failure mode. Since the risk estimator in GRA-HRE separately deals with the basic events representing human error modes such as control failure, wrong object, omission, wrong action, etc., we can recognize their relative importance comparing with other types of mechanical failures. Other specialties in GRA-HRE came from the combined application of fault tree analysis and turbine cycle simulation. The previous study suggested that we would use the fault tree analysis with the top events designated by system's malfunction such as 'feedwater system failure' to develop the risk estimator. However, this approach could not clearly provide the path of propagation of human errors, and it was difficult to present the failure logics in some cases. In order to overcome these bottlenecks, the paper is going to propose the modified idea to setup top events and to explain how to make use of turbine cycle simulation to complete the fault trees in a cooperative manner

Studies on Mineralogy, Micromorphology and Relationships of

African Journals Online (AJOL)

ced b y Sa b in et G a tew a. y u n d er licen ce gra n ted b y th e P u b lish er (d a ted. 2012) ... Acrisols), Itogoro (Eutric G leysols, .... '43. 19. 38. 0.59. 9.4. 7.5. 1.3. 0.61. 10.64. 0.56. 42.3. 2.8. 31.1. 91. Ck3 ..... ified by Soil Survey Staff (1990) for.

Journal of Genetics | Indian Academy of Sciences

Indian Academy of Sciences (India)

Plant vacuolar Na+/H+ antiporters play important roles in cellular ion homeostasis,vacuolar pH regulation and sequestration of Na+ ions into the vacuole. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na+/H+ transporting activity. In this study, we truncated the ...

Metal Tolerance Protein 8 Mediates Manganese Homeostasis and Iron Reallocation during Seed Development and Germination

Czech Academy of Sciences Publication Activity Database

Eroglu, S.; Giehl, R.F.H.; Meier, B.; Takahashi, M.; Terada, Y.; Ignatyev, K.; Andresen, Elisa; Küpper, Hendrik; Peiter, E.; von Wiren, N.

2017-01-01

Roč. 174, č. 3 (2017), s. 1633-1647 ISSN 0032-0889 Institutional support: RVO:60077344 Keywords : diffusion facilitator family * arabidopsis-thaliana * x-ray * vacuolar manganese Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 6.456, year: 2016

Optimizing the static-dynamic performance of the body-in-white using a modified non-dominated sorting genetic algorithm coupled with grey relational analysis

Science.gov (United States)

Wang, Dengfeng; Cai, Kefang

2018-04-01

This article presents a hybrid method combining a modified non-dominated sorting genetic algorithm (MNSGA-II) with grey relational analysis (GRA) to improve the static-dynamic performance of a body-in-white (BIW). First, an implicit parametric model of the BIW was built using SFE-CONCEPT software, and then the validity of the implicit parametric model was verified by physical testing. Eight shape design variables were defined for BIW beam structures based on the implicit parametric technology. Subsequently, MNSGA-II was used to determine the optimal combination of the design parameters that can improve the bending stiffness, torsion stiffness and low-order natural frequencies of the BIW without considerable increase in the mass. A set of non-dominated solutions was then obtained in the multi-objective optimization design. Finally, the grey entropy theory and GRA were applied to rank all non-dominated solutions from best to worst to determine the best trade-off solution. The comparison between the GRA and the technique for order of preference by similarity to ideal solution (TOPSIS) illustrated the reliability and rationality of GRA. Moreover, the effectiveness of the hybrid method was verified by the optimal results such that the bending stiffness, torsion stiffness, first order bending and first order torsion natural frequency were improved by 5.46%, 9.30%, 7.32% and 5.73%, respectively, with the mass of the BIW increasing by 1.30%.

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.

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

Directory of Open Access Journals (Sweden)

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.

A 16-kilodalton lipoprotein of the outer membrane of Serpulina (Treponema) hyodysenteriae.

Science.gov (United States)

Thomas, W; Sellwood, R; Lysons, R J

1992-08-01

Serpulina (Treponema) hyodysenteriae P18A and VS1 were extracted by using the detergent Triton X-114 and separated into detergent and aqueous phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis confirmed that a membrane-associated 16-kDa antigen was hydrophobic, since it was found in the detergent phase. A 45-kDa antigen partitioned into the aqueous phase, suggesting that it was hydrophilic and may be of periplasmic origin. When spirochetes were grown in the presence of [3H]palmitic acid, a predominant 16-kDa antigen was labeled; from the results of immunoprecipitation experiments, this antigen appeared to be the same as that recognized by both polyclonal and monoclonal antisera to a previously described 16-kDa antigen. This antigen was proteinase K sensitive and was not a component of the lipopolysaccharide, which, although [3H]palmitate labeled, was resistant to proteinase K digestion. The most probable explanation is that the 16-kDa antigen is a membrane-associated, surface-exposed, immunodominant lipoprotein.

Fermentative Degradation of Polyethylene Glycol by a Strictly Anaerobic, Gram-Negative, Nonsporeforming Bacterium, Pelobacter venetianus sp. nov

OpenAIRE

1983-01-01

The synthetic polyether polyethylene glycol (PEG) with a molecular weight of 20,000 was anaerobically degraded in enrichment cultures inoculated with mud of limnic and marine origins. Three strains (Gra PEG 1, Gra PEG 2, and Ko PEG 2) of rod-shaped, gram-negative, nonsporeforming, strictly anaerobic bacteria were isolated in mineral medium with PEG as the sole source of carbon and energy. All strains degraded dimers, oligomers, and polymers of PEG up to a molecular weight of 20,000 completely...

Chemical Mixing Model and K-Th-Ti Systematics and HED Meteorites for the Dawn Mission

Science.gov (United States)

Usui, T.; McSween, H. Y., Jr.; Mittlefehldt, D. W.; Prettyman, T. H.

2009-01-01

The Dawn mission will explore 4 Vesta, a large differentiated asteroid believed to be the parent body of the howardite, eucrite and diogenite (HED) meteorite suite. The Dawn spacecraft carries a gamma-ray and neutron detector (GRaND), which will measure the abundances of selected elements on the surface of Vesta. This study provides ways to leverage the large geochemical database on HED meteorites as a tool for interpreting chemical analyses by GRaND of mapped units on the surface of Vesta.

Diagnosis of pulmonary infection with Toxoplasma gondii in immunocompromised HIV-positive patients by real-time PCR

DEFF Research Database (Denmark)

Petersen, E.; Edvinsson, B.; Lundgren, Bettina

2006-01-01

. In positive samples, the genotype of the parasite was determined by sequence analysis of the GRA6 gene. Positive results were achieved for 2% (7/332) of the samples tested. Genotyping was possible in two samples and revealed GRA6 type II T. gondii. PCR for detecting T. gondii in BAL samples should...... be performed in all immunosuppressed HIV-positive patients with symptoms of a systemic infection of unknown etiology. Trimethoprim-sulfamethoxazole prophylaxis does not exclude concomitant infection with T. gondii....

Recent developments in high-resolution global altimetric gravity field modeling

DEFF Research Database (Denmark)

Andersen, Ole Baltazar; Knudsen, Per; Berry, P. A .M.

2010-01-01

older gravity fields show accuracy improvement of the order of 20-40% due to a combination of retracking, enhanced processing, and the use of the new EGM2008 geoid model. In coastal and polar regions, accuracy improved in many places by 40-50% (or more) compared with older global marine gravity fields.......In recent years, dedicated effort has been made to improve high-resolution global marine gravity fields. One new global field is the Danish National Space Center (DNSC) 1-minute grid called DNSC08GRA, released in 2008. DNSC08GRA was derived from double-retracked satellite altimetry, mainly from...... the ERS-1 geodetic mission data, augmented with new retracked GEOSAT data which have significantly enhanced the range and hence the gravity field accuracy. DNSC08GRA is the first high-resolution global gravity field to cover the entire Arctic Ocean all the way to the North Pole. Comparisons with other...

Hole Quality Assessment in Drilling of Glass Microballoon/Epoxy Syntactic Foams

Science.gov (United States)

Ashrith, H. S.; Doddamani, Mrityunjay; Gaitonde, Vinayak; Gupta, Nikhil

2018-05-01

Syntactic foams reinforced with glass microballoons are used as alternatives for conventional materials in structural application of aircrafts and automobiles due to their unique properties such as light weight, high compressive strength, and low moisture absorption. Drilling is the most commonly used process of making holes for assembling structural components. In the present investigation, grey relation analysis (GRA) is used to optimize cutting speed, feed, drill diameter, and filler content to minimize cylindricity, circularity error, and damage factor. Experiments based on full factorial design are conducted using a vertical computer numerical control machine and tungsten carbide twist drills. GRA reveals that a combination of lower cutting speed, filler content, and drill diameter produces a good quality hole at optimum intermediate feed in drilling syntactic foams composites. GRA also shows that the drill diameter has a significant effect on the hole quality. Furthermore, damage on the hole exit side is analyzed using a scanning electron microscope.

Automated Systems for Safeguarding and Accountancy of Stored Nuclear Material (for proceedings of ESARDA 21st Annual Meeting, Sevilla, Spain May 4-6, 1999

International Nuclear Information System (INIS)

Baldwin, K.M.; Bell, Z.W.; Dunigan, J.J.; Gaby, J.E.; Hickerson, T.W.; Lawson, R.L.; Miller, M.A.; Mooney, L.R.; Pickett, C.A.; Seiber, L.E.; Younkin, J.R.

1999-01-01

Oak Ridge has developed several sensor systems that are capable of providing unattended monitoring of the physical and/or assigned attributes associated with stored nuclear materials. These systems include the Continuous Automated Vault Inventory System (CAVISTM), SmartShelfTM, and the ReflectoActive Seal System TM. Each of these systems can be implemented independently or may be integrated with existing systems through the Graphical Facility Information Center or GraFICTM software package. GraFICTM is a versatile software package designed to operate in a distributed computing environment. GraFICTM can monitor and report all item and facility activity from the various sensors and systems to an unlimited number of authorized remote clients through a common interface. The software also contains an Intelligent Facility Management (lFM) package that helps storage facility managers with space planning, records management, item location, and variety of other facility specific needs. Results and details from several system deployments will be described, along with the specific features and possible uses of each system

Two showy traits, scent emission and pigmentation, are finely coregulated by the MYB transcription factor PH4 in petunia flowers.

Science.gov (United States)

Cna'ani, Alon; Spitzer-Rimon, Ben; Ravid, Jasmin; Farhi, Moran; Masci, Tania; Aravena-Calvo, Javiera; Ovadis, Marianna; Vainstein, Alexander

2015-11-01

The mechanism underlying the emission of phenylpropanoid volatiles is poorly understood. Here, we reveal the involvement of PH4, a petunia MYB-R2R3 transcription factor previously studied for its role in vacuolar acidification, in floral volatile emission. We used the virus-induced gene silencing (VIGS) approach to knock down PH4 expression in petunia, measured volatile emission and internal pool sizes by GC-MS, and analyzed transcript abundances of scent-related phenylpropanoid genes in flowers. Silencing of PH4 resulted in a marked decrease in floral phenylpropanoid volatile emission, with a concurrent increase in internal pool levels. Expression of scent-related phenylpropanoid genes was not affected. To identify putative scent-related targets of PH4, we silenced PH5, a tonoplast-localized H(+) -ATPase that maintains vacuolar pH homeostasis. Suppression of PH5 did not yield the reduced-emission phenotype, suggesting that PH4 does not operate in the context of floral scent through regulation of vacuolar pH. We conclude that PH4 is a key floral regulator that integrates volatile production and emission processes and interconnects two essential floral traits - color and scent. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Fructan active enzymes (FAZY) activities and biosynthesis of fructooligosaccharides in the vacuoles of Agave tequilana Weber Blue variety plants of different age.

Science.gov (United States)

Mellado-Mojica, Erika; González de la Vara, Luis E; López, Mercedes G

2017-02-01

Biosynthesis of agave fructans occurs in mesontle vacuoles which showed fluctuations in FAZY activities and synthesized a diverse spectrum of fructooligosaccharide isomers. Agave tequilana Weber Blue variety is an important agronomic crop in Mexico. Fructan metabolism in A. tequilana exhibits changes in fructan content, type, degree of polymerization (DP), and molecular structure. Specific activities of vacuolar fructan active enzymes (FAZY) in A. tequilana plants of different age and the biosynthesis of fructooligosaccharides (FOSs) were analyzed in this work. Vacuoles from mesontle (stem) protoplasts were isolated and collected from 2- to 7-year-old plants. For the first time, agave fructans were identified in the vacuolar content by HPAEC-PAD. Several FAZY activities (1-SST, 6-SFT, 6G-FFT, 1-FFT, and FEH) with fluctuations according to the plant age were found in protein vacuolar extracts. Among vacuolar FAZY, 1-SST activities appeared in all plant developmental stages, as well as 1-FFT and FEH activities. The enzymes 6G-FFT and 6-SST showed only minimal activities. Lowest and highest FAZY activities were found in 2- and 6-year-old plants, respectively. Synthesized products (FOS) were analyzed by TLC and HPAEC-PAD. Vacuolar FAZYs yielded large FOS isomers diversity, being 7-year-old plants the ones that synthesized a greater variety of fructans with different DP, linkages, and molecular structures. Based on the above, we are proposing a model for the FAZY activities constituting the FOS biosynthetic pathways in Agave tequilana Weber Blue variety.

Electrografting and morphological studies of chemical vapour deposition grown graphene sheets modified by electroreduction of aryldiazonium salts

International Nuclear Information System (INIS)

Mooste, Marek; Kibena, Elo; Kozlova, Jekaterina; Marandi, Margus; Matisen, Leonard; Niilisk, Ahti; Sammelselg, Väino; Tammeveski, Kaido

2015-01-01

Highlights: • CVD-grown graphene sheets were electrografted with various aryldiazonium salts • Redox grafting was applied to form thick nitrophenyl films • The reduction of the released radicals was in evidence during the redox grafting • Multilayer formation on CVD graphene was confirmed by XPS and AFM measurements • Thickness of different aryl layers on CVD graphene varied from few to 30 nm - Abstract: This work focuses on investigating the electrografting of chemical vapour deposition (CVD) graphene electrodes grown onto Ni foil (Ni/Gra) with different diazonium salts (including azobenzene diazonium tetrafluoroborate, Fast Garnet GBC sulphate salt, Fast Black K salt, 4-bromobenzene diazonium tetrafluoroborate and 4-nitrobenzenediazonium tetrafluoroborate). Various grafting conditions (e.g. “normal” electrografting in the narrow potential range and redox grafting in the wider potential range) were used. The electrochemical grafting behaviour was similar for all diazonium compounds used, except for the 4-nitrobenzenediazonium tetrafluoroborate when redox grafting was applied. The X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and Raman spectroscopy results confirmed the presence of the corresponding aryl layers on Ni/Gra surfaces. The formation of multilayers on Ni/Gra substrates was in evidence since the thickness of different aryl layers varied from few to 30 nm depending on the modification procedures as well as the diazonium compounds used and the XPS analysis revealed a peak at about 400 eV for all aryl-modified Ni/Gra samples suggesting the multilayer formation also through azo linkages

Optimization of process parameters through GRA, TOPSIS and RSA models

Directory of Open Access Journals (Sweden)

Suresh Nipanikar

2018-01-01

Full Text Available This article investigates the effect of cutting parameters on the surface roughness and flank wear during machining of titanium alloy Ti-6Al-4V ELI( Extra Low Interstitial in minimum quantity lubrication environment by using PVD TiAlN insert. Full factorial design of experiment was used for the machining 2 factors 3 levels and 2 factors 2 levels. Turning parameters studied were cutting speed (50, 65, 80 m/min, feed (0.08, 0.15, 0.2 mm/rev and depth of cut 0.5 mm constant. The results show that 44.61 % contribution of feed and 43.57 % contribution of cutting speed on surface roughness also 53.16 % contribution of cutting tool and 26.47 % contribution of cutting speed on tool flank wear. Grey relational analysis and TOPSIS method suggest the optimum combinations of machining parameters as cutting speed: 50 m/min, feed: 0.8 mm/rev., cutting tool: PVD TiAlN, cutting fluid: Palm oi

Tempus Actas – B 3 em Saúde Coletiva

Directory of Open Access Journals (Sweden)

Ana Valéria Machado MENDONCA

2015-10-01

Full Text Available Parece que foi ontem, quando lançamos em 2007, o volume 1, número 1 da Revista Tempus Actas em Saúde Coletiva, sob a coordenação da Unidade de Tecnologias da Informação e da Comunicação em Saúde do Núcleo de Estudos em Saúde Pública da Universidade de Brasília. Hoje, passados oito anos, a jovem revista científica, nascida no Centro-Oeste do país, tem ocupado cada vez mais espaços de reconhecimento no Brasil e no mundo, graças ao trabalho hercúleo de sua equipe de colaboradores(as, por sua disciplina e atenção aos novos tempos da comunicação científica que regem as produções de elevado rigor científico.

Položaj nacionalnih i vjerskih manjina u Bosni i Hercegovini

OpenAIRE

Kukić, Slavo

2002-01-01

Autor analizira rezultate empirijskog istraživanja “Položaj nacionalnih i vjerskih manjina u Bosni i Hercegovini”. Istraživanje je pokazalo da se u svoje predratne domove najsporije vraćaju građani srpske nacionalnosti. Spremnost na povratak iskazuje preko sedamdeset posto građana BiH. Što se tiče ljudskih prava, preko polovice stanovništva smatra da su u BiH ugrožena temeljna ljudska prava. Autor smatra da je rat doveo do velikih demografskih pokreta stanovništva te je utjecao na radikalno p...

AS "LETRAS" DO ESPÍRITO: S. INÁCIO INTÉRPRETE DA VIDA CRISTÃ

Directory of Open Access Journals (Sweden)

Ulpiano Várquez

1991-01-01

Full Text Available O argumento e ao mesmo tempo o resumo de minha palestra nesta noite é uma convicção: a vida cristã, a vida dos cristãos, se manifesta e se compreende graças ao testemunho das pessoas que viveram ou tentam viver o cristianismo radicalmente; graças às pessoas cuja vida só é inteligível por Jesus Cristo e, assim, nos mostram como é que de fato Cristo vive em nós; como é que de fato nós vivemos em Cristo.

Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

Science.gov (United States)

Sarkar, Mitul; Koland, John G

2016-01-01

The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

Optimisation of laser welding parameters for welding of P92 material using Taguchi based grey relational analysis

Directory of Open Access Journals (Sweden)

Shanmugarajan B.

2016-08-01

Full Text Available Creep strength enhanced ferritic (CSEF steels are used in advanced power plant systems for high temperature applications. P92 (Cr–W–Mo–V steel, classified under CSEF steels, is a candidate material for piping, tubing, etc., in ultra-super critical and advanced ultra-super critical boiler applications. In the present work, laser welding process has been optimised for P92 material by using Taguchi based grey relational analysis (GRA. Bead on plate (BOP trials were carried out using a 3.5 kW diffusion cooled slab CO2 laser by varying laser power, welding speed and focal position. The optimum parameters have been derived by considering the responses such as depth of penetration, weld width and heat affected zone (HAZ width. Analysis of variance (ANOVA has been used to analyse the effect of different parameters on the responses. Based on ANOVA, laser power of 3 kW, welding speed of 1 m/min and focal plane at −4 mm have evolved as optimised set of parameters. The responses of the optimised parameters obtained using the GRA have been verified experimentally and found to closely correlate with the predicted value.

Photolabeling of tonoplast from sugar beet cell suspensions by [3H]-MIA, an inhibitor of the vacuolar Na+/H+ antiport

International Nuclear Information System (INIS)

Barkla, B.J.; Blumwald, E.

1990-01-01

A radiolabeled amiloride analog, [ 3 H]-MIA, was used for equilibrium binding studies and photolabeling of purified tonoplast vesicles. Scatchard analysis revealed a high affinity binding component with a K 4 of 1.4 μM which is closely related to constants of inhibition obtained for Na + -dependent H + efflux (5.9 μM) and pH-dependent 22 Na + influx (2.5 μM). This suggests that the high affinity component represents a class of sites associated with the Na + /H + antiport. Photolabeling of tonoplast with [ 3 H]-MIA in the presence of amiloride revealed the presence of two classes of receptors with distinct affinities for MIA, possibly representing the Na + /H + antiport and the Na + channel. In order to identify these receptors, amiloride analogues specific for the Na + /H + antiport or the Na + channel are being used to protect differentially against labeling of tonoplast proteins by photo-irradiation of [ 3 H]-MIA

Transferability of hydrological models and ensemble averaging methods between contrasting climatic periods

Science.gov (United States)

Broderick, Ciaran; Matthews, Tom; Wilby, Robert L.; Bastola, Satish; Murphy, Conor

2016-10-01

Understanding hydrological model predictive capabilities under contrasting climate conditions enables more robust decision making. Using Differential Split Sample Testing (DSST), we analyze the performance of six hydrological models for 37 Irish catchments under climate conditions unlike those used for model training. Additionally, we consider four ensemble averaging techniques when examining interperiod transferability. DSST is conducted using 2/3 year noncontinuous blocks of (i) the wettest/driest years on record based on precipitation totals and (ii) years with a more/less pronounced seasonal precipitation regime. Model transferability between contrasting regimes was found to vary depending on the testing scenario, catchment, and evaluation criteria considered. As expected, the ensemble average outperformed most individual ensemble members. However, averaging techniques differed considerably in the number of times they surpassed the best individual model member. Bayesian Model Averaging (BMA) and the Granger-Ramanathan Averaging (GRA) method were found to outperform the simple arithmetic mean (SAM) and Akaike Information Criteria Averaging (AICA). Here GRA performed better than the best individual model in 51%-86% of cases (according to the Nash-Sutcliffe criterion). When assessing model predictive skill under climate change conditions we recommend (i) setting up DSST to select the best available analogues of expected annual mean and seasonal climate conditions; (ii) applying multiple performance criteria; (iii) testing transferability using a diverse set of catchments; and (iv) using a multimodel ensemble in conjunction with an appropriate averaging technique. Given the computational efficiency and performance of GRA relative to BMA, the former is recommended as the preferred ensemble averaging technique for climate assessment.

MAVS dimer is a crucial signaling component of innate immunity and the target of hepatitis C virus NS3/4A protease.

Science.gov (United States)

Baril, Martin; Racine, Marie-Eve; Penin, François; Lamarre, Daniel

2009-02-01

The mitochondrial antiviral signaling (MAVS) protein plays a central role in innate antiviral immunity. Upon recognition of a virus, intracellular receptors of the RIG-I-like helicase family interact with MAVS to trigger a signaling cascade. In this study, we investigate the requirement of the MAVS structure for enabling its signaling by structure-function analyses and resonance energy transfer approaches in live cells. We now report the essential role of the MAVS oligomer in signal transduction and map the transmembrane domain as the main determinant of dimerization. A combination of mutagenesis and computational methods identified a cluster of residues making favorable van der Waals interactions at the MAVS dimer interface. We also correlated the activation of IRF3 and NF-kappaB with MAVS oligomerization rather than its mitochondrial localization. Finally, we demonstrated that MAVS oligomerization is disrupted upon expression of HCV NS3/4A protease, suggesting a mechanism for the loss of antiviral signaling. Altogether, our data suggest that the MAVS oligomer is essential in the formation of a multiprotein membrane-associated signaling complex and enables downstream activation of IRF3 and NF-kappaB in antiviral innate immunity.

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

Science.gov (United States)

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

Avaliação da produção de batata-doce em função de níveis de adubação orgânica = Evaluation of sweet potato yield in function of organic fertilization levels

Directory of Open Access Journals (Sweden)

João Felinto dos Santos

2010-10-01

Full Text Available O presente trabalho avaliou o efeito da adubação orgânica na produçãototal de batata-doce, por meio de um experimento realizado no período de julho a novembro de 2007, na Estação Experimental de Lagoa Seca, PB, Brasil. O delineamento experimental foi em blocos casualizados com cinco tratamentos (0, 3, 6, 9 e 12 t ha-1 de cama-de-aviário, em quatro repetições. A parcela foi composta por quatro leiras de 3,6 m de comprimento, espaçados de 0,80 m entre leiras e de 0,30 m entre plantas, sendo colhidas as duas fileiras centrais. As variáveis estudadas foram: produção total, raízes de tamanho graúdas e miúdas de batata-doce. A produção de raízes graúdas, miúdas e total por planta (14,20; 3,84 e 18,03 t ha-1 de batata-doce responderam de forma linear e positiva às doses crescentes de cama-de-aviário. Aadubação com cama-de-aviário aumentou a produção de raízes graúdas, miúdas e total de batata-doce.The present study evaluated the effect of organic fertilization inthe total production of sweet potato. The experiment was performed in the period between July and November 2007, at the Lagoa Seca Experimental Station, Paraíba, Brazil. The experimental design was randomized blocks with five treatments (0, 3, 6, 9 and 12 t ha-1, in four replications. The plot consisted of four 3.6 m long ridges, spaced of 0.80 m between ridges and 0.30 m between plants; the two central ridges were harvested. The variables studied were: total yield, large and small sweet potato roots. The production of large and small roots, total yield per plant (14.20, 3.84 and 18.03 t ha-1of sweet potato, responded positively in a linear fashion to increasing doses of poultry litter. The fertilization with poultry litter increased the yield of large, small and total roots of sweet potato.

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.

The impact on quality of life of dialysis patients with renal insufficiency

OpenAIRE

Dąbrowska-Bender M; Dykowska G; Żuk W; Milewska M; Staniszewska A

2018-01-01

Marta Dąbrowska-Bender,1 Grażyna Dykowska,2 Wioletta Żuk,3 Magdalena Milewska,1 Anna Staniszewska4 1Department of Clinical Dietetics, Medical University of Warsaw, Warsaw, Poland; 2Department of Public Health, Medical University of Warsaw, Warsaw, Poland; 3Department of Surgery, Transplantation and Extracorporeal Surgery, Medical University of Warsaw, Warsaw, Poland; 4Department of Experimental and Clinical Pharmacology, Medical University of Warsaw, Warsaw, Poland Aim: The aim of the st...

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.

Examining the Determinants of China’s Inward FDI Using Grey Matrix Relational Analysis Model

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Hang JIANG

2017-12-01

Full Text Available Grey relational analysis (GRA model is an important part of grey system theory, which is used to ascertain the relational grade between an influential factor and the major behavior factor. Most of GRA models are mainly applied to the field in which the behavior factor and influential factor are the cross-sectional or time series data in a given system. However, owing to the panel data contains plenty information including individual and time characteristics, the traditional GRA model cannot be applied to panel data analysis. To overcome this drawback, the grey matrix relational analysis model is applied to measure the similarity of panel data from two dimensions of individual and time on the basis of the definition of the matrix sequence of a discrete data sequence. This paper examines the determinants of inward foreign direct investment (IFDI in China using grey matrix relational analysis model. The study finds that the GDP per capita, enrollment of regular institutions of higher education, and internal expenditure on R&D are the key factors of IFDI.

Evaluation of banks’ commercial credit applications using the analytic hierarchy process and Grey relational analysis: A comparison between public and private banks

Directory of Open Access Journals (Sweden)

Zeliha Kaygısız Ertuğ

2015-08-01

Full Text Available The purpose of this study is to develop an evaluation model that considers the quantitative and qualitative criteria for the appropriate selection of firms demanding commercial credit for both public and private banks. In this paper, the authors propose an integrated model that combines the Analytic Hierarchy Process (AHP and Grey Relational Analysis (GRA into a single evaluation model. The model is illustrated with a case study on bank experts to demonstrate the effectiveness of this integrated method for four firms that applied for a commercial loan. In this study, AHP is applied to determine the weight of the criteria, and GRA is performed to determine the most appropriate firm. The results of this study indicate that, whereas firm morality and news criteria are the main criteria with the highest priority, sale and marketing constructions are the main criteria with the lowest priorities for both public and private banks. In addition, according to the results of GRA, the most appropriate firm for a public bank is Firm 1, and the most appropriate firm for a private bank is Firm 2.

Dicty_cDB: CHE174 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available stallinum mRNA for putative vacuolar ATPase subunit 100 kDa subunit (vpha-a gene). 46 1e-10 4 CV164410 |CV164410.1 rsmsx...54 8e-09 3 CV172465 |CV172465.1 rsmsxlre_008717.z1.scf cDNA Library of Salvia mil

A chimeric repressor of petunia PH4 R2R3-MYB family transcription factor generates margined flowers in torenia.

Science.gov (United States)

Kasajima, Ichiro; Sasaki, Katsutomo

2016-05-03

The development of new phenotypes is key to the commercial development of the main floricultural species and cultivars. Important new phenotypes include features such as multiple-flowers, color variations, increased flower size, new petal shapes, variegation and distinctive petal margin colourations. Although their commercial use is not yet common, the transgenic technologies provide a potentially rapid means of generating interesting new phenotypes. In this report, we construct 5 vectors which we expected to change the color of the flower anthocyanins, from purple to blue, regulating vacuolar pH. When these constructs were transformed into purple torenia, we unexpectedly recovered some genotypes having slightly margined petals. These transgenic lines expressed a chimeric repressor of the petunia PhPH4 gene under the control of Cauliflower mosaic virus 35 S RNA promoter. PhPH4 is an R2R3-type MYB transcription factor. The transgenic lines lacked pigmentation in the petal margin cells both on the adaxial and abaxial surfaces. Expressions of Flavanone 3-hydroxylase (F3H), Flavonoid 3'-hydroxylase (F3'H) and Flavonoid 3'5'-hydroxylase (F3'5'H) genes were reduced in the margins of these transgenic lines, suggesting an inhibitory effect of PhPH4 repressor on anthocyanin synthesis.

A Systems Biology Approach Reveals Differences in the Dynamics of Colonization and Degradation of Grass vs. Hay by Rumen Microbes with Minor Effects of Vitamin E Supplementation

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Alejandro Belanche

2017-08-01

Full Text Available Increasing the efficiency of utilization of fresh and preserved forage is a key target for ruminant science. Vitamin E is often used as additive to improve product quality but its impact of the rumen function is unknown. This study investigated the successional microbial colonization of ryegrass (GRA vs. ryegrass hay (HAY in presence of zero or 50 IU/d supplementary vitamin E, using a rumen simulation technique. A holistic approach was used to link the dynamics of feed degradation with the structure of the liquid-associated (LAB and solid-associated bacteria (SAB. Results showed that forage colonization by SAB was a tri-phasic process highly affected by the forage conservation method: Early colonization (0–2 h after feeding by rumen microbes was 2× faster for GRA than HAY diets and dominated by Lactobacillus and Prevotella which promoted increased levels of lactate (+56% and ammonia (+18%. HAY diets had lower DM degradation (-72% during this interval being Streptococcus particularly abundant. During secondary colonization (4–8 h the SAB community increased in size and decreased in diversity as the secondary colonizers took over (Pseudobutyrivibrio promoting the biggest differences in the metabolomics profile between diets. Secondary colonization was 3× slower for HAY vs. GRA diets, but this delay was compensated by a greater bacterial diversity (+197 OTUs and network complexity resulting in similar feed degradations. Tertiary colonization (>8 h consisted of a slowdown in the colonization process and simplification of the bacterial network. This slowdown was less evident for HAY diets which had higher levels of tertiary colonizers (Butyrivibrio and Ruminococcus and may explain the higher DM degradation (+52% during this interval. The LAB community was particularly active during the early fermentation of GRA and during the late fermentation for HAY diets indicating that the availability of nutrients in the liquid phase reflects the dynamics

Isolation of Arachis hypogaea Na /H antiporter and its expression ...

African Journals Online (AJOL)

Jane

2011-10-24

Oct 24, 2011 ... /H. + antiporter and its expression analysis under salt stress ... Xinguo Li2,3*. 1College of Life Science, Linyi University, Linyi 276005, China. ... gene was isolated from peanut (Arachis hypogaea) in the present work. The full-length ..... balance (Rausch et al., 1996), with the vacuolar Na+/H+ antiporter gene ...

Sum of ratios of products forα-μ random variables in wireless multihop relaying and multiple scattering

KAUST Repository

Wang, Kezhi; Wang, Tian; Chen, Yunfei; Alouini, Mohamed-Slim

2014-01-01

The sum of ratios of products of independent 2642 2642α-μ random variables (RVs) is approximated by using the Generalized Gamma ratio approximation (GGRA) with Gamma ratio approximation (GRA) as a special case. The proposed approximation is used to calculate the outage probability of the equal gain combining (EGC) or maximum ratio combining (MRC) receivers for wireless multihop relaying or multiple scattering systems considering interferences. Numerical results show that the newly derived approximation works very well verified by the simulation, while GRA has a slightly worse performance than GGRA when outage probability is below 0.1 but with a more simplified form.

Preparation of Composited Graphene/PEDOT:PSS Film for Its Possible Application in Graphene-based Organic Solar Cells

Institute of Scientific and Technical Information of China (English)

YU Yue; LI Meicheng; CHU Lihua; YU Hakki; Wodtke A M; ZHAO Yan; ZHANG Zhongmo

2015-01-01

The interface between graphene and organic layers is a key factor responsible for the performance of gra-phene-based organic solar cells (OSCs). In this paper, we focus on coating PEDOT:PSS onto the surface of graphene. We demonstrate two approaches, applying UV/Ozone treatment on graphene and modifying PEDOT:PSS with Zonyl, to get a PEDOT:PSS well-coated graphene film . Our results prove that both methods can be effective to solve the interface issue between graphene and PEDOT: PSS. Thereby it shows a positive application of the composited gra-phene/PEDOT:PSS film on graphene-based OSCs.

Sum of ratios of products forα-μ random variables in wireless multihop relaying and multiple scattering

KAUST Repository

Wang, Kezhi

2014-09-01

The sum of ratios of products of independent 2642 2642α-μ random variables (RVs) is approximated by using the Generalized Gamma ratio approximation (GGRA) with Gamma ratio approximation (GRA) as a special case. The proposed approximation is used to calculate the outage probability of the equal gain combining (EGC) or maximum ratio combining (MRC) receivers for wireless multihop relaying or multiple scattering systems considering interferences. Numerical results show that the newly derived approximation works very well verified by the simulation, while GRA has a slightly worse performance than GGRA when outage probability is below 0.1 but with a more simplified form.

Läget i berggrummet : En kvalitativ undersökning av inverkande faktorer i arbetsmiljön på en byggarbetsplats i ett bergrum

OpenAIRE

Miladi, Lubna

2014-01-01

Strävan efter att minska risker för arbetsskador och uppnå en god arbetsmiljö är betydelsefull idag inte minst på byggarbetsplatser.   Syftet med examensarbetet var att, utifrån kartläggning av arbetsmiljön på en byggarbetsplats, belysa några faktorer som har inverkan på arbetsmiljön. Magisteruppsatsen har begränsats till att undersöka arbetsmiljön på en byggarbetsplats i ett bergrum. Några organisatoriska faktorer som säkerhetskultur, attityder, kunskap samt fysiska faktorer som belysning, b...

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

Delivering of Proteins to the Plant Vacuole—An Update

Directory of Open Access Journals (Sweden)

Cláudia Pereira

2014-05-01

Full Text Available Trafficking of soluble cargo to the vacuole is far from being a closed issue as it can occur by different routes and involve different intermediates. The textbook view of proteins being sorted at the post-Golgi level to the lytic vacuole via the pre-vacuole or to the protein storage vacuole mediated by dense vesicles is now challenged as novel routes are being disclosed and vacuoles with intermediate characteristics described. The identification of Vacuolar Sorting Determinants is a key signature to understand protein trafficking to the vacuole. Despite the long established vacuolar signals, some others have been described in the last few years, with different properties that can be specific for some cells or some types of vacuoles. There are also reports of proteins having two different vacuolar signals and their significance is questionable: a way to increase the efficiency of the sorting or different sorting depending on the protein roles in a specific context? Along came the idea of differential vacuolar sorting, suggesting a possible specialization of the trafficking pathways according to the type of cell and specific needs. In this review, we show the recent advances in the field and focus on different aspects of protein trafficking to the vacuoles.

Studies on the membrane-associated proteolytic activities of Escherichia coli

International Nuclear Information System (INIS)

Palmer, S.M.

1986-01-01

The membrane fraction contains three proteolytic activities which can be resolved from whole membrane detergent extracts by DEAE-cellulose chromatography. The first two eluting activities have been previously reported as protease V and protease IV. These two enzymes were further purified by gel permeation HPLC. Protease V has a M. W. of 31,000 in SDS-PAGE gels. Protease IV has a M. W. of 62,000 and exists in two distinct isoforms of pl ≅ 6.7 and 6.9. The third enzyme eluting from the DEAE-cellulose column was further purified by affinity chromatography on Benzamidine-Sepharose 6B. This enzyme, referred to herein as protease VI, is a 43 kdal protein which has not been previously characterized. Protease VI was sensitive to inhibition by the serine protease inhibitors phenylmethylsulfony fluoride (PMSF), diisopropylfluorophosphate (DFP) and p-amino-benzamidine (PAB). Additionally, this enzyme exhibited maximal activity at pH ≅ 8.0. Incubation of whole membrane preparations with [ 3 H] DFP resulted in 11 specific proteins acquiring the radioactive label, included in this group of proteins were proteases IV, V, and VI. Several of the DFP-reactive proteins were also shown to bind [ 125 I] ampicillin

Multi-Responses Optimization Of Edm Sinking Process Of Aisi D2 Tool Steel Using Taguchi Grey–Fuzzy Method

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Bobby Oedy Pramoedyo Soepangkat

2014-12-01

Full Text Available Rough machining with Electro Discharge Machining (EDM process gives a large Material Removal Rate (MRR and high Surface Roughness (SR, while finish machining gives low SR and very slow MRR. In this study, Taguchi method coupled with Grey Relational Analysis (GRA and fuzzy logic has been applied for optimization of multiple performance characteristics. The EDM machining parameters (gap voltage, pulse current, on time and duty factor are optimized with considerations of multiple performance characteristics, i.e., MRR and SR. The quality characteristic of MRR is larger-is-better, while the quality characteristic of SR is smaller-is-better. Based on Taguchi method, an L18 mixed-orthogonal array is selected for the experiments. By using the combination of GRA and fuzzy logic, the optimization of complicated multiple performance characteristics was transformed into the optimization of a single response performance index. The most significant machining parameters which affect the multiple performance characteristics were gapvoltage and pulse current. Experimental results have also shown that machining performance characteristics of EDM process can be improved effectively through the combination of Taguchi method, GRA and fuzzy logic.

Silene noctiflora L., present in the flora of Kosovo and Metohija (Serbia

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Prodanović, D.

2016-12-01

Full Text Available The genus Silene (family Caryophyllaceae comprises more than 700 species and it is one of the larger genera of the World’s flora. Studying the flora of the plain part of Kosovo and Metohija, in the village of Gračanica, on the eastern rim of the Kosovo basin (approximately 10 km to the south of Priština, near the Gračanka stream, we identified a weed and ruderal species, Silene noctiflora. The part of the stream bank is urbanised and turned into a quay. The quay is regularly maintained implying that the banks are regularly weeded and the riverbeds are cleaned as the Typha latifolia species rapidly grows; it may be the reason why this species was not immediately detected and appropriately identified. Except for the afore mentioned locality, the species was also reported on the neglected arable fields and tilths, around the unkempt orchards, along the new tarmac road between Gračanica and Laplje Selo, on the locality called Labura. Silene noctiflora is not mentioned for the region of Kosovo and Metohija in the new and revised edition of the book Flora of Serbia 2.

O Semantema

Directory of Open Access Journals (Sweden)

Jorge Antunes

2000-10-01

Full Text Available O autor vem, durante os últimos anos, desenvolvendo pesquisa no domínio da correspondência e das conversões biunívocas entre sons e imagens gráficas. As primeiras etapas dessa pesquisa, que envolveram a utilização do sistema UPIC, se diversificaram nos campos da composição musical, da análise musical de obras eletroacústicas e na pedagogia da escuta musical. Nova ramificação da pesquisa aqui se delineia. As experiências com as "representações gráficas" permitiram a constatação de que "o ato de ouvir música está sempre acompanhado de grafismos inconscientes que a mente e o intelecto praticam em espaços imaginários". Nesse trabalho são apresentados os resultados da pesquisa, que se caracterizam como identificações de unidades semânticas, de conotação gráfico-espacial-temporal, baseadas na emoção.

Grey relational analysis for evaluating the effects of different rates of wine lees-derived biochar application on a plant-soil system with multi-metal contamination.

Science.gov (United States)

Xu, Min; Zhu, Qihong; Wu, Jun; He, Yan; Yang, Gang; Zhang, Xiaohong; Li, Li; Yu, Xiaoyu; Peng, Hong; Wang, Lilin

2018-03-01

In this study, grey relational analysis (GRA) was used to investigate the effects of different application rates of wine lees-derived biochar on a plant-soil system with multi-metal contamination. A pot experiment was conducted to determine rice growth in multi-metal-contaminated soil amended with samples of wine lees-derived biochar, and 47 indicators (including soil properties, microbial activity, and plant physiology) were selected as evaluation indexes to assess the plant-soil system. The results indicated that higher wine lees-derived biochar application rates (2% W/W) were favorable for soil fertility, the bioconcentration factor (BF), and the mobility factor (MF, %) (with the exception of Cr, Zn, and Hg), but an application of 1% produced the highest plant growth, enzymatic activities, and bacterial diversity. The richness of the bacterial communities was reduced in the soil amended with the wine lees-derived biochar. According to the GRA assessment, the 1% application rate of wine lees-derived biochar was more suitable for restoring the holistic plant-soil system than were the application rates of 0, 0.5, and 2% (W/W). Furthermore, this study shows that GRA is a useful method for evaluating plant-soil systems.

Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions

Czech Academy of Sciences Publication Activity Database

Svobodová, Kateřina; Mikesková, Hana; Petráčková, Denisa

2013-01-01

Roč. 97, č. 24 (2013), s. 10263-10273 ISSN 0175-7598 R&D Projects: GA TA ČR TE01020218 Institutional support: RVO:61388971 Keywords : Fungal microsomes * Cytochrome P450 * Biodegradation Subject RIV: EE - Microbiology, Virology Impact factor: 3.811, year: 2013

Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

Science.gov (United States)

Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

2015-01-01

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

Renovação interior do ser humano e sua deificação/santificação

Directory of Open Access Journals (Sweden)

Kunrath, Pedro Alberto

2007-01-01

Full Text Available A graça, mais que estudá-la, deve-se vivê-la. Mas é necessário compreendê-la. Primeiro, o modo da passagem da situação de pecado para a situação da graça: a justificação; e segundo, o resultado dessa passagem: a justiça. Por que Deus conserva o homem na existência, quando este está condenado à morte? Conserva-o para justificá-lo. Visa uma transformação, uma renovação da vida; significa que visa eliminar o pecado e tomar o homem realmente justo

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

International Nuclear Information System (INIS)

Towner, Jonathan S.; Brown, David M.; Nguyen, Joseph H.C.; Semler, Bert L.

2003-01-01

In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required

Plasma proteins of Barbus holubi and Clarias gariepinus

African Journals Online (AJOL)

The supernatant fluids were subsequently examined by gel electrophoresis. Control experiments showed that the 3-hour exposure as such had no effect on the electrophoretogram. Zoo/oglca A!ricana 9(2):185-193 (1974). 185. R ep ro d u ced b y Sa b in et G a tew a. y u n d er licen ce gra n ted b y th e P u b lish er (d a ted.

OBSERVATIONS ON SOCIAL ORGANIZATION OF SPRINGBOK ...

African Journals Online (AJOL)

Ansell (1968) there are two larger races, A.m. hofmeyri. which occurs in the southern part of. South West Africa and Botswana, and A.m. angolensis, which occurs in the northern part of South West Africa and Angola. ZoologiCQ AjricQnQ l3(1): l/5-122 (1978). R ep rod u ced b y Sa b in et G a tew a. y u n d er licen ce gra n ted.

National Dam Safety Program. No Name 165 (Crystal Lake Dam) (MO 10580), Missouri - Kansas City Basin, Jackson County, Missouri. Phase I Inspection Report.

Science.gov (United States)

1978-07-01

For NTIS GRA&I DTIC TAB >0 Unannounced [D Just ification- D T C ELECTE By Distribution/ NOV 20 1981 Avail and/orS Availabilit CodesD=-Dist Spca D NO...Hutton, Engineering Geologist. Impoundment of water began in 1970. h. Normal Operating Procedure. Normal rainfall, runoff, transpir- ation, and...evaporation all combine to maintain a relatively stable water surface elevation. 1.3 PERTINENT DATA a. Drainage Area - 9,900 acres of which approximately 15

Forage mass and stocking rate of elephant grass pastures managed under agroecological and conventional systems

OpenAIRE

Clair Jorge Olivo; Carlos Alberto Agnolin; Priscila Flôres Aguirre; Cláudia Marques de Bem; Tiago Luís da Ros de Araújo; Michelle Schalemberg Diehl; Gilmar Roberto Meinerz

2014-01-01

The objective was to evaluate elephant grass (Pennisetum purpureum Schum.) pastures, under the agroecological and conventional systems, as forage mass and stocking rate. In the agroecological system, the elephant grass was established in rows spaced by 3.0 m from each other. During the cool season ryegrass (Lolium multiflorum Lam.) was established between these rows, which allowed the development of spontaneous growth species during the warm season. In the conventional system the elephant gra...

Repeated tender point injections of granisetron alleviate chronic myofascial pain--a randomized, controlled, double-blinded trial.

Science.gov (United States)

Christidis, Nikolaos; Omrani, Shahin; Fredriksson, Lars; Gjelset, Mattias; Louca, Sofia; Hedenberg-Magnusson, Britt; Ernberg, Malin

2015-01-01

Serotonin (5-HT) mediates pain by peripheral 5-HT3-receptors. Results from a few studies indicate that intramuscular injections of 5-HT3-antagonists may reduce musculoskeletal pain. The aim of this study was to investigate if repeated intramuscular tender-point injections of the 5-HT3-antagonist granisetron alleviate pain in patients with myofascial temporomandibular disorders (M-TMD). This prospective, randomized, controlled, double blind, parallel-arm trial (RCT) was carried out during at two centers in Stockholm, Sweden. The randomization was performed by a researcher who did not participate in data collection with an internet-based application ( www.randomization.com ). 40 patients with a diagnose of M-TMD according to the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) were randomized to receive repeated injections, one week apart, with either granisetron (GRA; 3 mg) or isotonic saline as control (CTR). The median weekly pain intensities decreased significantly at all follow-ups (1-, 2-, 6-months) in the GRA-group (Friedman test; P  0.075). The numbers needed to treat (NNT) were 4 at the 1- and 6-month follow-ups, and 3.3 at the 2-month follow-up in favor of granisetron. Repeated intramuscular tender-point injections with granisetron provide a new pharmacological treatment possibility for myofascial pain patients with repeated intramuscular tender-point injections with the serotonin type 3 antagonist granisetron. It showed a clinically relevant pain reducing effect in the temporomandibular region, both in a short- and long-term aspect. European Clinical Trials Database 2005-006042-41 as well as at Clinical Trials NCT02230371 .

A New Crucial Protein Interaction Element That Targets the Adenovirus E4-ORF1 Oncoprotein to Membrane Vesicles▿

OpenAIRE

Chung, Sang-Hyuk; Frese, Kristopher K.; Weiss, Robert S.; Prasad, B. V. Venkataram; Javier, Ronald T.

2007-01-01

Human adenovirus type 9 exclusively elicits mammary tumors in experimental animals, and the primary oncogenic determinant of this virus is the E4-ORF1 oncogene, as opposed to the well-known E1A and E1B oncogenes. The tumorigenic potential of E4-ORF1, as well as its ability to oncogenically stimulate phosphatidylinositol 3-kinase (PI3K), depends on a carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with several different membrane-associated cellular PDZ proteins, inc...

SC*994C>T causes the Sc(null) phenotype in Pacific Islanders and successful transfusion of Sc3+ blood to a patient with anti-Sc3.

Science.gov (United States)

Reid, Marion E; Hue-Roye, Kim; Velliquette, Randall W; Larimore, Kathleen; Moscarelli, Sue; Ohswaldt, Nicolas; Lomas-Francis, Christine

2013-01-01

Antigens in the SC blood group system are expressed by the human erythrocyte membrane-associated protein (ERMAP).Two molecular bases have been reported for the Sc,un phenotype:SC*307del2 and SC*994C>T. We report our investigation of the molecular background of five Sc,n1 individuals from the Pacific Islands and describe the successful transfusion of Sc3+ blood to a patient with anti-Sc3 in her plasma. SC (ERMAP) exons 2,3, and 12 and their flanking intronic regions were analyzed. TheSC*994C>T change introduces a restriction enzyme cleavage site for Tsp45I, and polymerase chain reaction (PCR) products from exon 12 were subjected to this PCR-restriction fragment length polymorphism (RFLP) assay. The five samples had the variant SC*994T/T. One sample, from a first cousin of one Marshallese proband, was heterozygous for SC*1514C/T (in the 3' untranslated region); the other four samples were SC*1514C/C(consensus sequence). Samples from white donors (n = 100) and African American donors (n = 99) were tested using the Tsp45IPCR-RFLP assay; all gave a banding pattern that was consistent with the SC*994C/C consensus sequence. In all five samples,our analyses showed homozygosity for the nonsense nucleotide change SC*994C>Tin an allele carrying the nucleotide associated with SLd. Further investigation determined that one of the probands reported previously with the SC*994C>T change was from the Marshall Islands (which form part of the Micronesian Pacific Islands) and the other was from an unspecified location within the large collection of Pacific Islands. Taken together, the five known probands with the SC*994C>T silencing nucleotide change were from the Pacific Islands.

Glycoprotein A repetitions predominant (GARP) positively regulates transforming growth factor (TGF) β3 and is essential for mouse palatogenesis.

Science.gov (United States)

Wu, Bill X; Li, Anqi; Lei, Liming; Kaneko, Satoshi; Wallace, Caroline; Li, Xue; Li, Zihai

2017-11-03

Glycoprotein A repetitions predominant (GARP) (encoded by the Lrrc32 gene) plays important roles in cell-surface docking and activation of TGFβ. However, GARP's role in organ development in mammalian systems is unclear. To determine the function of GARP in vivo , we generated a GARP KO mouse model. Unexpectedly, the GARP KO mice died within 24 h after birth and exhibited defective palatogenesis without apparent abnormalities in other major organs. Furthermore, we observed decreased apoptosis and SMAD2 phosphorylation in the medial edge epithelial cells of the palatal shelf of GARP KO embryos at embryonic day 14.5 (E14.5), indicating a defect in the TGFβ signaling pathway in the GARP-null developing palates. Of note, the failure to develop the secondary palate and concurrent reduction of SMAD phosphorylation without other defects in GARP KO mice phenocopied TGFβ3 KO mice, although GARP has not been suggested previously to interact with TGFβ3. We found that GARP and TGFβ3 co-localize in medial edge epithelial cells at E14.5. In vitro studies confirmed that GARP and TGFβ3 directly interact and that GARP is indispensable for the surface expression of membrane-associated latent TGFβ3. Our findings indicate that GARP is essential for normal morphogenesis of the palate and demonstrate that GARP plays a crucial role in regulating TGFβ3 signaling during embryogenesis. In conclusion, we have uncovered a novel function of GARP in positively regulating TGFβ3 activation and function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Przemoc domowa = Domestic violence

OpenAIRE

Łepecka-Klusek, Celina; Pawłowska-Muc, Agnieszka Konstancja; Pilewska-Kozak, Anna Bogusława; Stadnicka, Grażyna; Pałucka, Klaudia

2015-01-01

Łepecka-Klusek Celina, Pawłowska-Muc Agnieszka Konstancja, Pilewska‑Kozak Anna Bogusława, Stadnicka Grażyna, Pałucka Klaudia. Przemoc domowa = Domestic violence. Journal of Education, Health and Sport. 2015;5(6):169-182. ISSN 2391-8306. DOI 10.5281/zenodo.18420 http://ojs.ukw.edu.pl/index.php/johs/article/view/2015%3B5%286%29%3A169-182 https://pbn.nauka.gov.pl/works/564476 http://dx.doi.org/10.5281/zenodo.18420 Formerly Journal of Health Sciences. ISSN 1429-9623 / 2300-665X. A...

The effect of antipsychotic drugs on nonspecific inflammation markers in the first episode of schizophrenia

Directory of Open Access Journals (Sweden)

Stefanović Vesna

2015-01-01

Full Text Available Background/Aim. Immune system disorder, including inflammation, takes a significant place when considering still unclear etiology of schizophrenia. The aim of this study was to determine the blood levels of nonspecific inflammation markers in the first episode of schizophrenia and their relation to the therapy response. Methods. In this study we determined the blood levels of nonspecific inflammation markers: white blood cells count (WBC, C-reactive protein (CRP, erythrocytes sedimentation rate (ESR and the elements of differential white blood cell counts (or the leukocyte formula: granulocytes (Gra, lymphocytes (Lym and monocytes (Mon, in the first episode of schizofrenia, in 78 patients hospitalized at the Clinic for Psychiatric Disorders “Dr Laza Lazarević” in Belgrade. The levels were measured at admission to the clinic, as well as after 4 weeks of antipsychotic treatment. The Positive and negative syndrome scale for schizophrenia (PANSS was applied to measure the severity of psychopathology and response to the treatment. Results. During the first episode of schizophrenia, before initiation of antipsychotic treatment, the frequency of abnormal values was high (≥ 25% of the patients for the following non-specific inflammation markers: WBC, CRP, ESR and Gra, in the leukocyte formula, but dropped after 4 weeks of antipsychotic treatment at the level of high statistical significance for WBC and Gra (p < 0.001. The ESR remained unchanged in as many as 50% of the patients even after 4-week antipsychotic treatment, at the level of statistical significance in the non-responders compared to the responders (p = 0.045. Conclusion. The obtained results indicate that in the first episode of schizophrenia the blood levels of non-specific inflammation markers (WBS, CRP, ESR and Gra from the leukocyte formula were high in the subpopulation of patients with the tendency towards normalization of inflammation parameters after a 4-week antipsychotic

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.

GraMi: Generalized Frequent Pattern Mining in a Single Large Graph

KAUST Repository

Saeedy, Mohammed El

2011-11-01

Mining frequent subgraphs is an important operation on graphs. Most existing work assumes a database of many small graphs, but modern applications, such as social networks, citation graphs or protein-protein interaction in bioinformatics, are modeled as a single large graph. Interesting interactions in such applications may be transitive (e.g., friend of a friend). Existing methods, however, search for frequent isomorphic (i.e., exact match) subgraphs and cannot discover many useful patterns. In this paper the authors propose GRAMI, a framework that generalizes frequent subgraph mining in a large single graph. GRAMI discovers frequent patterns. A pattern is a graph where edges are generalized to distance-constrained paths. Depending on the definition of the distance function, many instantiations of the framework are possible. Both directed and undirected graphs, as well as multiple labels per vertex, are supported. The authors developed an efficient implementation of the framework that models the frequency resolution phase as a constraint satisfaction problem, in order to avoid the costly enumeration of all instances of each pattern in the graph. The authors also implemented CGRAMI, a version that supports structural and semantic constraints; and AGRAMI, an approximate version that supports very large graphs. The experiments on real data demonstrate that the authors framework is up to 3 orders of magnitude faster and discovers more interesting patterns than existing approaches.

Origin of the color of Cv. rhapsody in blue rose and some other so-called "blue" roses.

Science.gov (United States)

Gonnet, Jean-François

2003-08-13

Flowers of the rose cultivar Rhapsody in Blue display unusual colors, changing as they age, from a vivid red-purple to a lighter and duller purple, which are based on tonalities corresponding to hue angles between 340 and 320 degrees in the CIELAB scale. Unexpectedly, the chemical basis of these colors is among the simplest, featuring cyanin (cyanidin 3,5-di-O-glucoside), the most frequent anthocyanin in flowers, as the sole pigment and quercetin kaempferol glycosides as copigments at a relatively low copigment/pigment ratio (about 3/1), which usually produces magenta or red shades in roses. This color shift to bluer shades is coupled with the progressive accumulation of cyanin into vacuolar anthocyanic inclusions (AVIs), the occurrence of which increases as the petals grow older. In addition to the normal lambda(max) of cyanin at approximately 545 nm, the transmission spectra of live petals and of epidermal cells exhibit a second lambda(max) in the 620-625 nm range, the relative importance increasing with the presence of AVIs. In petals of fully opened flowers, the only pigmented structures in the vacuoles of epidermal cells are AVIs; their intense and massive absorption in the 520-640 nm area produces a much darker and bluer color than measured for the vacuolar solution present at the very first opening stage. Cyanin is probably "trapped" into AVIs at higher concentrations than would be possible in a vacuolar solution and in quinonoidal form, appearing purple-blue because of additional absorption in the 580-630 nm area. Quite similar pigmentation features were found in very ancient rose cultivars (cv. L'Evêque or Bleu Magenta), also displaying this type of so-called "blue" color.

Higher education, Graduate unemployment, Poverty and Economic growth in Tunisia, 1990-2013

Directory of Open Access Journals (Sweden)

Haifa Mefteh

2016-07-01

Full Text Available This paper examines the relationship between economic growth, higher education, unemployment and poverty using properties of time series variables while applying the Ordinary Least Squares (OLS method. Our study thus contributes to the existing literature by giving the first integrated approach to examine the four way linkages in the Tunisian background over the period 1990-2013. This paper holds that higher education can impact unemployment and graduate unemployment causes poverty which would affect economic growth. Our empirical results show that there is bi-directional causal relationship between per capita gross domestic product (GDP and poverty rate (POV and also between Number of graduate students (GRA and School enrollment tertiary education (ENR besides unidirectional causal relationship which running from Number of graduate students to Unemployment with tertiary education (UNP, from Higher education expenditure (EXP to poverty rate and from Unemployment with tertiary education to poverty rate. Our empirical results also verified the existence of positive effect of ENR, GRA and POV on economic growth, while, UNP and EXP have negative determining influence on economic growth with only GRA statistically significant. These empirical insights are of particular interest for the policy makers as they help build sound economic policies to sustain economic development and improve the higher educational quality.

Nondestructive continuous physical property measurements of core samples recovered from hole B, Taiwan Chelungpu-Fault Drilling Project

Science.gov (United States)

Hirono, Tetsuro; Yeh, En-Chao; Lin, Weiren; Sone, Hiroki; Mishima, Toshiaki; Soh, Wonn; Hashimoto, Yoshitaka; Matsubayashi, Osamu; Aoike, Kan; Ito, Hisao; Kinoshita, Masataka; Murayama, Masafumi; Song, Sheng-Rong; Ma, Kuo-Fong; Hung, Jih-Hao; Wang, Chien-Ying; Tsai, Yi-Ben; Kondo, Tomomi; Nishimura, Masahiro; Moriya, Soichi; Tanaka, Tomoyuki; Fujiki, Toru; Maeda, Lena; Muraki, Hiroaki; Kuramoto, Toshikatsu; Sugiyama, Kazuhiro; Sugawara, Toshikatsu

2007-07-01

The Taiwan Chelungpu-Fault Drilling Project was undertaken in 2002 to investigate the faulting mechanism of the 1999 Mw 7.6 Taiwan Chi-Chi earthquake. Hole B penetrated the Chelungpu fault, and core samples were recovered from between 948.42- and 1352.60-m depth. Three major zones, designated FZB1136 (fault zone at 1136-m depth in hole B), FZB1194, and FZB1243, were recognized in the core samples as active fault zones within the Chelungpu fault. Nondestructive continuous physical property measurements, conducted on all core samples, revealed that the three major fault zones were characterized by low gamma ray attenuation (GRA) densities and high magnetic susceptibilities. Extensive fracturing and cracks within the fault zones and/or loss of atoms with high atomic number, but not a measurement artifact, might have caused the low GRA densities, whereas the high magnetic susceptibility values might have resulted from the formation of magnetic minerals from paramagnetic minerals by frictional heating. Minor fault zones were characterized by low GRA densities and no change in magnetic susceptibility, and the latter may indicate that these minor zones experienced relatively low frictional heating. Magnetic susceptibility in a fault zone may be key to the determination that frictional heating occurred during an earthquake on the fault.

Perianth bottom-specific blue color development in Tulip cv. Murasakizuisho requires ferric ions.

Science.gov (United States)

Shoji, Kazuaki; Miki, Naoko; Nakajima, Noriyuki; Momonoi, Kazumi; Kato, Chiharu; Yoshida, Kumi

2007-02-01

The entire flower of Tulipa gesneriana cv. Murasakizuisho is purple, except the bottom, which is blue. To elucidate the mechanism of the different color development in the same petal, we prepared protoplasts from the purple and blue epidermal regions and measured the flavonoid composition by HPLC, the vacuolar pH by a proton-selective microelectrode, and element contents by the inductively coupled plasma (ICP) method. Chemical analyses revealed that the anthocyanin and flavonol compositions in both purple and blue colored protoplasts were the same; delphinidin 3-O-rutinoside (1) and major three flavonol glycosides, manghaslin (2), rutin (3) and mauritianin (4). The vacuolar pH values of the purple and blue protoplasts were 5.5 and 5.6, respectively, without any significant difference. However, the Fe(3+) content in the blue protoplast was approximately 9.5 mM, which was 25 times higher than that in the purple protoplasts. We could reproduce the purple solution by mixing 1 with two equimolar concentrations of flavonol with lambda(vismax) = 539 nm, which was identical to that of the purple protoplasts. Furthermore, addition of Fe(3+) to the mixture of 1-4 gave the blue solution with lambda(vismax) = 615 nm identical to that of the blue protoplasts. We have established that Fe(3+) is essential for blue color development in the tulip.

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.

Investigating the role of viral integral membrane proteins in promoting the assembly of nepovirus and comovirus replication factories

Directory of Open Access Journals (Sweden)

Helene eSanfacon

2013-01-01

Full Text Available Formation of plant virus membrane-associated replication factories requires the association of viral replication proteins and viral RNA with intracellular membranes, the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex. Many viruses encode integral membrane proteins that act as anchors for the replication complex. These hydrophobic proteins contain trans-membrane domains and/or amphipathic helices that associate with the membrane and modify its structure. The comovirus Co-Pro and NTP-binding (NTB, putative helicase proteins and the cognate nepovirus X2 and NTB proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2B, 2C and 3A membrane proteins. The identification of membrane-association domains and analysis of the membrane topology of these proteins is discussed. The evidence suggesting that these proteins have the ability to induce membrane proliferation, alter the structure and integrity of intracellular membranes and modulate the induction of symptoms in infected plants is also reviewed. Finally, areas of research that need further investigation are highlighted.

Potentiation of the generation of reactive oxidants by human phagocytes during exposure to benoxaprofen and ultraviolet radiation in vitro

Energy Technology Data Exchange (ETDEWEB)

Anderson, R.; Eftychis, H.A.

1986-09-01

The effects of ultraviolet (UV) radiation on the spontaneous membrane-associated oxidative metabolism of human polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (MNL), co-incubated in the presence and absence of the non-steroidal, anti-inflammatory drug (NSAID) benoxaprofen at various concentrations, were investigated in vitro. Assays of superoxide generation and luminol-enhanced chemiluminescence (CL) were used to detect the production of reactive oxidants by PMNL and MNL. The pro-oxidative effects of benoxaprofen and UV radiation alone and in combination are dependent on intact phagocyte membrane-associated oxidative metabolism. It is postulated that the pro-oxidative interactions which occur between human phagocytes, benoxaprofen and ultraviolet radiation cause the dermatological side-effects of benoxaprofen.

Dicty_cDB: SSE424 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 2 CE00209 PROBABLE VACUOLAR ATP SYNTHASE SUBUNIT D ;, mRNA sequence. 68 2e-07 1 CD147641 |CD147641.1 ML1-000...7T-V150-B09-U.B ML1-0007 Schistosoma mansoni cDNA clone ML1-0007T-V150-B09.B, mRNA sequence. 56 2e-07 3 CD147632 |CD147

Molecular characterization, light-dependent expression, and cellular localization of a host vacuolar-type H+-ATPase (VHA) subunit A in the giant clam, Tridacna squamosa, indicate the involvement of the host VHA in the uptake of inorganic carbon and its supply to the symbiotic zooxanthellae.

Science.gov (United States)

Ip, Yuen K; Hiong, Kum C; Lim, Leon J Y; Choo, Celine Y L; Boo, Mel V; Wong, Wai P; Neo, Mei L; Chew, Shit F

2018-06-15

The giant clam, Tridacna squamosa, represents a clam-zooxanthellae association. In light, the host clam and the symbiotic zooxanthellae conduct light-enhanced calcification and photosynthesis, respectively. We had cloned the cDNA coding sequence of a Vacuolar-type Proton ATPase (VHA) subunit A, ATP6V1A, from T. squamosa, whereby the VHA is an electrogenic transporter that actively 'pumps' H + out of the cell. The ATP6V1A of T. squamosa comprised 1866 bp, encoding a protein of 622 amino acids and 69.9 kDa, and had a host-origin. Its gene expression was strong in the ctenidium and the colorful outer mantle, but weak in the whitish inner mantle, corroborating a previous proposition that VHA might have a trivial role in light-enhanced calcification. Light exposure led to significant increases in the gene and protein expression levels of ATP6V1A/ATP6V1A in the ctenidium and the outer mantle. In the ctenidium, the ATP6V1A was localized in the apical epithelia of the filaments and tertiary water channels, indicating that the VHA could participate in the increased excretion of H + produced during light-enhanced calcification. Additionally, the excreted H + would augment HCO 3 - dehydration in the external medium and facilitate the uptake of CO 2 by the ctenidium during insolation. In the outer mantle, the ATP6V1A was detected in intracellular vesicles in a type of cells, presumably iridocytes, surrounding the zooxanthellal tubules, and in the apical epithelium of zooxanthellal tubules. Hence, the host VHA could participate in the transfer of inorganic carbon from the hemolymph to the luminal fluid of the tubules by increasing the supply of H + for the dehydration of HCO 3 - to CO 2 during insolation to benefit the photosynthesizing zooxanthellae. Copyright © 2018 Elsevier B.V. All rights reserved.

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

International Nuclear Information System (INIS)

Walker, G.; Bourguignon, L.Y.

1990-01-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Energy Technology Data Exchange (ETDEWEB)

Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

1990-08-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

S1PR3 Signaling Drives Bacterial Killing and Is Required for Survival in Bacterial Sepsis.

Science.gov (United States)

Hou, JinChao; Chen, QiXing; Wu, XiaoLiang; Zhao, DongYan; Reuveni, Hadas; Licht, Tamar; Xu, MengLong; Hu, Hu; Hoeft, Andreas; Ben-Sasson, Shmuel A; Shu, Qiang; Fang, XiangMing

2017-12-15

Efficient elimination of pathogenic bacteria is a critical determinant in the outcome of sepsis. Sphingosine-1-phosphate receptor 3 (S1PR3) mediates multiple aspects of the inflammatory response during sepsis, but whether S1PR3 signaling is necessary for eliminating the invading pathogens remains unknown. To investigate the role of S1PR3 in antibacterial immunity during sepsis. Loss- and gain-of-function experiments were performed using cell and murine models. S1PR3 levels were determined in patients with sepsis and healthy volunteers. S1PR3 protein levels were up-regulated in macrophages upon bacterial stimulation. S1pr3 -/- mice showed increased mortality and increased bacterial burden in multiple models of sepsis. The transfer of wild-type bone marrow-derived macrophages rescued S1pr3 -/- mice from lethal sepsis. S1PR3-overexpressing macrophages further ameliorated the mortality rate of sepsis. Loss of S1PR3 led to markedly decreased bacterial killing in macrophages. Enhancing endogenous S1PR3 activity using a peptide agonist potentiated the macrophage bactericidal function and improved survival rates in multiple models of sepsis. Mechanically, the reactive oxygen species levels were decreased and phagosome maturation was delayed in S1pr3 -/- macrophages due to impaired recruitment of vacuolar protein-sorting 34 to the phagosomes. In addition, S1RP3 expression levels were elevated in monocytes from patients with sepsis. Higher levels of monocytic S1PR3 were associated with efficient intracellular bactericidal activity, better immune status, and preferable outcomes. S1PR3 signaling drives bacterial killing and is essential for survival in bacterial sepsis. Interventions targeting S1PR3 signaling could have translational implications for manipulating the innate immune response to combat pathogens.

Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells▿

Science.gov (United States)

Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

2008-01-01

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions. PMID:18684830

Different types of nsP3-containing protein complexes in Sindbis virus-infected cells.

Science.gov (United States)

Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

2008-10-01

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

Overview of Considerations in Assessing the Biomass Potential of Army Installations.

Science.gov (United States)

1981-08-01

techniques of harvest- ing biomass, (3) the feasibility of military development of energy planta - tions, (4) the economic feasibility of using biomass, (5...harvest system. 18 1. S. Goldstein, D. L. Holley, and E. L. Deal, "Economic Aspects of Low Gra de Ha rdwood Ut iIi za t ion ," Fore-s-t- Pro-du-cts J...equipment and techniques, (d) an initial assessment of potential silvicultural and ecologi- cal implications, and (e) an identification of managerial and

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

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

Science.gov (United States)

Gilmour, K M; Perry, S F; Bernier, N J; Henry, R P; Wood, C M

2001-01-01

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.

Prikazi

Directory of Open Access Journals (Sweden)

Drahomira Gavranović

2012-02-01

čina objavljivanja bavi se problemima do kojih dolazi ukoliko se obavlja istraživanje na nekom originalnom rukopisnom dokumentu. Autor navodi i na koje se sve probleme nailazi tijekom transkripcije notarskih zapisa, poput teškoća pri razrješavanju kratica, razlika između antičkog i srednjovjekovnog načina pisanja i sl. Posljednji rad u ovoj cjelini jest rad Z. Velagića pod naslovom Skriveni autor: pojavnosti autorskog “ja” na ranonovovjekovnim tiskanim naslovnicama. Autor analizira tri problema koji se vezuju uz autorstvo vjerskih djela tiskanih tijekom 18. stoljeća: je li autorovo ime dovoljno za pripisivanje autorstva toj osobi, zatim je li se na naslovnici bilježilo „pravo“ autorstvo te na kojim se još mjestima autor „obraćao“ čitateljima u svoje ime. Na kraju autor istražuje je li se knjiga koju danas držimo anonimnim djelom u doba kada je nastala također smatrala anonimnom. Autor uz rad donosi i naslovnice hrvatskih knjiga iz 18. stoljeća na kojima je provedeno istraživanje.Druga tematska cjelina ovoga zbornika nazvana je Stara knjiga kao predmet istraživanja i bibliografije (Old book as a topic for research and bibliography. Prvi rad u toj cjelini rad je autorice D. Sečić pod naslovom Knjižna baština u europskim i hrvatskim knjižnicama. Autorica je izradila pregled europskih knjižničnih fondova od humanizma do danas. Autorica se posebno osvrće na crkvene, dvorske i sveučilišne knjižnice koje sadrže iznimno bogate zbirke. Ovaj rad obuhvaća i analizu svih dostupnih mrežnih stranica te projekata koji se bave zbirkama stare i rijetke građe, čineći tako polazno mjesto za istraživanje svim budućim istraživačima nedovoljno istraženih europskih knjižničnih fondova. Velik dio rada autorica je posvetila analizi hrvatskih knjižnih fondova stare i rijetke građe, navodeći pritom i današnje stanje i otvorenost javnosti te upućujući na mogućnosti daljnjih istraživanja. Drugi rad u ovoj cjelini rad je autora E. Renharta pod

Identificação de Genótipos de Feijão-caupi Tolerantes a Acidez em um Latossolo Vermelho-Amarelo do Estado de Roraima = Identification of cowpea genotypes sensitivity to acidicy conditions in an Oxisol of Roraima State (Brazil.

Directory of Open Access Journals (Sweden)

Sandra Cátia Pereira Uchôa

2007-12-01

Full Text Available O presente trabalho foi realizado com o objetivo de identificar genótipos de feijão-caupi (Vigna unguiculata (L Walp., tolerantes à acidez. O trabalho foi realizado em casa de vegetação localizada no Campus do Cauamé, da Universidade Federal de Roraima, utilizando um Latossolo Vermelho-Amarelo de Boa Vista-RR. Foi adotado o delineamento experimental de Blocos Inteiramente Casualizados em esquema fatorial 2 x 5 x 10 com três repetições. Os tratamentos resultaram de duas formas de localização do calcário (localizada e não-localizada, 5 profundidades (0-5; 5-10; 10-15; 15-20; 20-25 cm e 10 genótipos de feijão-caupi(IT85D-3428-4-R2,4-HM; Apiaú; Hikari Graúdo; Pretinho Precoce 1; IT85D-3428-4-3-HP; USA; UFRR Grão Verde; BRS-Mazagão; Canapum e Sempre Verde. A unidade experimental foi constituída por 2dm3 de solo, dispostos em tubo de PVC com 25cm de altura,10cm de diâmetro e duas plantas de feijão. Os tubos foram confeccionados por meio da junção de 5 anéis com 5 cm cada, sendo que no anel inferior foi colocada uma placa delgada de isopor para permitir a acomodação do solo. Aos 40 dias após a emergência, as plantas foram coletadas, sendo determinada à produção de matéria seca das raízes e parte aérea. Os genótipos IT85D-3428-4-3-HP e UFRRGRÃO VERDE foram classificados como sendo de baixa tolerância, os genótipos USA, BRS-Mazagão, IT85D-3428-4-R2,4-HM e Sempre Verde, medianamente tolerantes e os genótipos Apiaú, Hikari Graúdo, Pretinho Precoce 1 e Canapum, tolerantes à acidez.= This research was carried out objecting to identify the sensibility of cowpea (Vigna unguiculata (L Walp genotypes to acidity conditions. The experiment was installed in a greenhouse located at Campus of Cauamé (Universidade Federal de Roraima in an oxisol of Roraima State (Brazil. The experimental design adopted was a completely andomized blocks in a factorial scheme 2 x 5 x 10 with three replicates. The terms of this factorial

The Effect of Herbicides on Hydrogen Peroxide Generation in Isolated Vacuoles of Red Beet Root (Beta vulgaris L.

Directory of Open Access Journals (Sweden)

E.V. Pradedova

2015-12-01

Full Text Available Influence of herbicides on the hydrogen peroxide generation in vacuolar extracts of red beet root (Beta vulgaris L. was investigated. Belonging to different chemical classes of herbicide compounds have been used. Herbicides differ from each other in the mechanism of effects on plants. Clopyralid (aromatic acid herbicide, derivative of picolinic acid and 2.4-D (phenoxyacetic herbicide, characterized by hormone-like effects, contributed to the formation of H2O2 in vacuolar extracts. Fluorodifen (nitrophenyl ether herbicide and diuron (urea herbicide also have increased contents H2O2. These compounds inhibit the electron transport, photosynthesis, and photorespiration in sensitive plants. Herbicidal effect of glyphosate (organophosphorus herbicide is due to the inhibition of amino acid synthesis in plant cells. Glyphosate did not affect the content of H2O2 in vacuolar extracts. Herbicide dependent H2O2-generation did not occur with oxidoreductase inhibitors, potassium cyanide and sodium azide. The results suggest that the formation of ROS in the vacuoles due to activity of oxidoreductases, which could interact with herbicides.

Research in electrohydrodynamics. Final report

International Nuclear Information System (INIS)

1973-01-01

An overview is presented in the form of publications for NASA research in electrohydrodynamics. Data cover basic electrohydrodynamics, management of liquids in zero gravity, continuum feedback control, and magnetohydrodynamics and ferrohydrodynamics. (GRA)

Basaltic scorias from Romania - complex building material us for concrete, glazing tiles, ceramic glazes, glass ceramics, mineral wool

Energy Technology Data Exchange (ETDEWEB)

Marica, S.; Cetean, V. [PROCEMA S.A., Bucharest (Romania)

2002-07-01

The most spectacular deposit of basaltic scoria from Romania is the Heghes Hill from Racos, locality situated in the central part of country. This deposit emerged as grains of various dimensions, as volcanic ash with specific porosity up to 30% and vacuolar basaltic rocks. All types of basaltic scorias have specific vacuolar appearance, red- brick or blackish - grey coloured, scoria textures and similar chemical composition with others basalts of the world. The physical and mechanical characteristics determined included the scorias in the Heghes Hill in the following categories : light rocks (2,98 g/ dmc), porous(11,04%), similar to expanded slag, slightly absorbing rocks (3,86%), with low compression strengths (1700 daN/cmp). Basaltic scoria from Heghes is a very good row material for the manufacture of concrete, for obtain decorative cutting tiles glazing with ceramic and basaltic glazes (up to 40%) varied the range of colours and for obtaining glass ceramic, mineral wool, crushing sand for road maintenance, heat -insulating bricks and shid -proof material. (orig.)

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.

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.

Importance of field-reversing ion ring formation in hot electron plasma

Energy Technology Data Exchange (ETDEWEB)

Ikuta, K.

1975-11-01

Formation of the field reversing ion ring in the mirror confined hot electron plasma may offer a device to confine the fusion plasma even under the restriction of the present technology. (Author) (GRA)

Microbial-based evaluation of anaerobic membrane bioreactors (AnMBRs) for the sustainable and efficient treatment of municipal wastewater

KAUST Repository

Harb, Moustapha

2017-01-01

in both aerobic and anaerobic MBR effluents by using molecularbased detection methods. The findings of this dissertation demonstrate that membrane-associated anaerobic digestion processes have significant potential to improve the sustainability

MBA1 encodes a mitochondrial membrane-associated protein required for biogenesis of the respiratory chain.

Science.gov (United States)

Rep, M; Grivell, L A

1996-06-17

The yeast MBA 1 gene (Multi-copy Bypass of AFG3) is one of three genes whose overexpression suppresses afg3-null and rca1-null mutations. Bypass of AFG3 and RCA1, whose products are essential for assembly of mitochondrial inner membrane enzyme complexes, suggests a related role for MBA1. The predicted translation product is a 30 kDa hydrophilic protein with a putative mitochondrial targeting sequence and no homology to any sequence in protein or EST databases. Gene disruption leads to a partial respiratory growth defect, which is more pronounced at temperatures above 30 degrees C. Concomitantly, amounts of cytochromes b and aa3 are reduced. A C-terminal c-myc-tagged MBA1 gene product is functional and is found associated with the mitochondrial inner membrane, from which it can he extracted by carbonate, but not by high salt. These observations give further support to a role of MBA1 in assembly of the respiratory chain.

Original Research Original Research

African Journals Online (AJOL)

RAGHAVENDRA

Twelve gra using simple random sampling technique and an En ... Science, Technology and Arts Research Journ. Sci. Technol. ... he quantitative data disclosed lower student scores, .... work on relating the title to students' lives, motivating,.

Explore the Most Potential Supplier’s Selection Determinants in Modern Supply Chain Management

Directory of Open Access Journals (Sweden)

Ming-Yuan Hsieh

2014-01-01

Full Text Available To increment the research reliability, validity, and representativeness, this study creatively cross-employed the factor analysis (FA and the grey relational analysis (GRA methods. The results of the 144 fully completed questionnaires are analyzed by FA and then these results were utilized in second questionnaires design of 15 experts. Furthermore, the results of these second questionnaires were further analyzed by GRA in order to explore the most potential supplier’s selection determinants in the modern supply chain management (MSCM. Beyond a series of measurements, the measured results have induced three contributive findings: (1 the empirical interviewed industrialists reported concern that suppliers have to provide a higher material yield rate and material delivery on time rate for the qualitative increment as well as a higher supplier’s gross margin ROI for the financial stabilization in MSCM; (2 the 15 experts concluded that material insurance rate is an important attribute to estimate risky assessments and the supplier’s gross margin ROI and warehouse operations cost as a percentage of sales are critical elements in the financial evaluations of potential suppliers; and (3 Supplier’s gross margin ROI, outbound freight cost as a percentage of sales, and material insurance rate are the three most decisive determinants in MSCM.

Dicty_cDB: SSD315 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available PROBABLE VACUOLAR ATP SYNTHASE SUBUNIT D ;, mRNA sequence. 68 2e-07 1 CD147641 |CD147641.1 ML1-0007T-V150-B...09-U.B ML1-0007 Schistosoma mansoni cDNA clone ML1-0007T-V150-B09.B, mRNA sequence. 56 3e-07 3 CD147...632 |CD147632.1 ML1-0007T-V150-A12-U.B ML1-0007 Schistosoma mansoni cDNA clone ML1-0007T-

Prevalence of Toxoplasma gondii infection in HIV-infected patients and food animals and direct genotyping of T. gondii isolates, Southern Ghana.

Science.gov (United States)

Pappoe, Faustina; Cheng, Weisheng; Wang, Lin; Li, Yuanling; Obiri-Yeboah, Dorcas; Nuvor, Samuel Victor; Ambachew, Henock; Hu, Xiaodong; Luo, Qingli; Chu, Deyong; Xu, Yuanhong; Shen, Jilong

2017-06-01

Toxoplasma gondii is of public health and veterinary importance causing severe diseases in immunocompromised individuals including HIV/AIDS patients and in congenital cases and animals. There is limited information on the epidemiology of T. gondii infection in humans, particularly HIV patients and food animals and the parasite genotypes in Ghana. A total of 394 HIV-infected patients from three hospitals were screened for T. gondii anti-IgG and IgM using ELISA. DNAs from blood samples of seropositve participants and 95 brain tissues of food animals were PCR assayed to detect Toxoplasma gra6. DNA positive samples were genotyped using multilocus nested polymerase chain reaction restriction fragment length polymorphism at 10 loci: sag1, alt.sag2, sag3, btub, gra6, l358, c22-8, c29-2, pk1, and apico. The overall seroprevalence was 74.37% (293/394). Toxoplasma DNAs were detected in 3.07% of the seropositive participants and 9.47% of the animals. Six of the human DNA positive samples were partly typed at sag3: 33.33, 50, and 16.67% isolates had type I, II, and III alleles, respectively. All nine isolates from food animals typed at nine loci except apico were atypical: six isolates were identical to ToxoDB #41 and #145, and one was identical to TgCkBrRj2 all identified in Brazil. The genotype of two isolates has not been reported previously and was named as TgCtGh1. T. gondii seroprevalence is high among the HIV-infected individuals with T. gondii circulating in Ghana being genetically diverse.

Estimation of glucose rate of appearance from cgs and subcutaneous insulin delivery in type 1 diabetes

KAUST Repository

Laleg-Kirati, Taous-Meriem; Al-Matouq, Ali Ahmed

2017-01-01

Method and System for providing estimates of Glucose Rate of Appearance from the intestine (GRA) using continuous glucose sensor measurements (CGS) taken from the subcutaneous of a diabetes patient and the amount of insulin administered

Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells

OpenAIRE

Walseng, Even; Furuta, Kazuyuki; Bosch, Berta; Weih, Karis A.; Matsuki, Yohei; Bakke, Oddmund; Ishido, Satoshi; Roche, Paul A.

2010-01-01

The expression and turnover of MHC class II-peptide complexes (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to activate CD4 T cells efficiently. The half-life of surface pMHC-II is significantly greater in activated (mature) DCs than in resting (immature) DCs, but the molecular mechanism leading to this difference remains unknown. We now show that ubiquitination of pMHC-II by the E3 ubiquitin ligase membrane-associated RING-CH 1 (March-I) regulates surface e...

Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching

DEFF Research Database (Denmark)

Adkins, Erika M; Samuvel, Devadoss J; Fog, Jacob U

2007-01-01

To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent...... protein) a diffusion coefficient (D) of approximately 3.6 x 10(-9) cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization...

Membrane mobility and microdomain association of the dopaminetransporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching

DEFF Research Database (Denmark)

Adkins, Erika; Samuvel, Devadoss; Fog, Jacob

2007-01-01

To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent...... protein) a diffusion coefficient (D) of ~3.6 × 10-9 cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization...

Arabidopsis CDS blastp result: AK105135 [KOME

Lifescience Database Archive (English)

Full Text Available AK105135 001-102-A05 At2g27170.1 structural maintenance of chromosomes (SMC) family protein similar to basem...ent membrane-associated chondroitin proteoglycan Bamacan [Rattus norvegicus] GI:178

On 4-critical t-perfect graphs

OpenAIRE

Benchetrit, Yohann

2016-01-01

It is an open question whether the chromatic number of $t$-perfect graphs is bounded by a constant. The largest known value for this parameter is 4, and the only example of a 4-critical $t$-perfect graph, due to Laurent and Seymour, is the complement of the line graph of the prism $\\Pi$ (a graph is 4-critical if it has chromatic number 4 and all its proper induced subgraphs are 3-colorable). In this paper, we show a new example of a 4-critical $t$-perfect graph: the complement of the line gra...

La Motivación y la Enseñanza de la Matemática en los estudiantes de los octavo, noveno y décimo grado de Educación General Básica de la escuela "Indoamérica", ubicada en la Parroquia Canchagua - Cantón Saquisilï - Provincia de Cotopaxi, en el año lectivo 2013 - 2014

OpenAIRE

Erazo Cajas, Alicia Beatriz

2015-01-01

1. Problema de investigación. --2. Marco Teórico. --3. Metodología del trabajo. --4. Análisis e interpretación de resultados. --5. Propuesta. --6. Conclusiones y Recomendaciones. El presente trabajo de investigación se basa en el uso de estrategias motivacionales para superar dificultades en el aprendizaje de la matemática. El objetivo general del proyecto fue establecer la incidencia de la motivación en la enseñanza de la matemática en los estudiantes de los octavos, novenos y décimos gra...

Proper and incorrect body posture in students from music schools

Directory of Open Access Journals (Sweden)

Roland Hadlich

2017-02-01

1. Wymuszona i długotrwała gra na wybranym instrumencie wpływa głownie na kąt zgięcia i wyprostu tułowia w płaszczyźnie strzałkowej. 2. Wykonawstwo na sześciu analizowanych instrumentach powoduje pogłębienie krzywizn fizjologicznych kręgosłupa: 3. Stwierdzone wielkości wybranych cech strzałkowych kręgosłupa u uczniów szkół muzycznych wymaga wprowadzenia postępowania profilaktyczno-korekcyjne.

Antarctic Meteorite Newsletter. Volume 20

Science.gov (United States)

Lindstrom, Marilyn M.; Satterwhite, Cecilia E.

1997-01-01

The availability of 116 new meteorites from the 1994-1996 collections is announced. There are 4 special chondrites, 2 carbonaceous chondrites, and 1 achondrite among the new meteorites. Also included is a redescription of Lodranite GRA95209.

Arabidopsis Intracellular NHX-Type Sodium-Proton Antiporters are Required for Seed Storage Protein Processing.

Science.gov (United States)

Ashnest, Joanne R; Huynh, Dung L; Dragwidge, Jonathan M; Ford, Brett A; Gendall, Anthony R

2015-11-01

The Arabidopsis intracellular sodium-proton exchanger (NHX) proteins AtNHX5 and AtNHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is poorly understood. Here we show that NHX5 and NHX6 are necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) technology that the C-terminal tail of NHX6 interacts with a component of Retromer, another component of the cell sorting machinery, and that this tail is critical for NHX6 activity. These findings demonstrate that NHX5 and NHX6 are important in processing and activity of vacuolar cargo, and suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Fermentative degradation of polyethylene glycol by a strictly anaerobic, gram-negative, nonsporeforming bacterium, Pelobacter venetianus sp. nov.

Science.gov (United States)

Schink, B; Stieb, M

1983-06-01

The synthetic polyether polyethylene glycol (PEG) with a molecular weight of 20,000 was anaerobically degraded in enrichment cultures inoculated with mud of limnic and marine origins. Three strains (Gra PEG 1, Gra PEG 2, and Ko PEG 2) of rod-shaped, gram-negative, nonsporeforming, strictly anaerobic bacteria were isolated in mineral medium with PEG as the sole source of carbon and energy. All strains degraded dimers, oligomers, and polymers of PEG up to a molecular weight of 20,000 completely by fermentation to nearly equal amounts of acetate and ethanol. The monomer ethylene glycol was not degraded. An ethylene glycol-fermenting anaerobe (strain Gra EG 12) isolated from the same enrichments was identified as Acetobacterium woodii. The PEG-fermenting strains did not excrete extracellular depolymerizing enzymes and were inhibited by ethylene glycol, probably owing to a blocking of the cellular uptake system. PEG, some PEG-containing nonionic detergents, 1,2-propanediol, 1,2-butanediol, glycerol, and acetoin were the only growth substrates utilized of a broad variety of sugars, organic acids, and alcohols. The isolates did not reduce sulfate, sulfur, thiosulfate, or nitrate and were independent of growth factors. In coculture with A. woodii or Methanospirillum hungatei, PEGs and ethanol were completely fermented to acetate (and methane). A marine isolate is described as the type strain of a new species, Pelobacter venetianus sp. nov. Its physiology and ecological significance, as well as the importance and possible mechanism of anaerobic polyether degradation, are discussed.

Molecular detection of Toxoplasma gondii in snakes.

Science.gov (United States)

Nasiri, Vahid; Teymurzadeh, Shohreh; Karimi, Gholamreza; Nasiri, Mehdi

2016-10-01

Toxoplasma gondii, an obligate intracellular protozoan parasite, is responsible for one of the most common zoonotic parasitic diseases in almost all warm-blooded vertebrates worldwide, and it is estimated that about one-third of the world human population is chronically infected with this parasite. Little is known about the circulation of T. gondii in snakes and this study for the first time aimed to evaluate the infection rates of snakes by this parasite by PCR methods. The brain of 68 Snakes, that were collected between May 2012 and September 2015 and died after the hold in captivity, under which they were kept for taking poisons, were examined for the presence of this parasite. DNA was extracted and Nested-PCR method was carried out with two of pairs of primers to detect the 344 bp fragment of T. gondii GRA6 gene. Five positive nested-PCR products were directly sequenced in the forward and reverse directions by Sequetech Company (Mountain View, CA). T. gondii GRA6 gene were detected from 55 (80.88%) of 68 snakes brains. Sequencing of the GRA6 gene revealed 98-100% of similarity with T. gondii sequences deposited in GenBank. To our knowledge, this is the first study of molecular detection of T. gondii in snakes and our findings show a higher frequency of this organism among them. Copyright © 2016 Elsevier Inc. All rights reserved.

Multi-response optimization of T300/epoxy prepreg tape-wound cylinder by grey relational analysis coupled with the response surface method

Science.gov (United States)

Kang, Chao; Shi, Yaoyao; He, Xiaodong; Yu, Tao; Deng, Bo; Zhang, Hongji; Sun, Pengcheng; Zhang, Wenbin

2017-09-01

This study investigates the multi-objective optimization of quality characteristics for a T300/epoxy prepreg tape-wound cylinder. The method integrates the Taguchi method, grey relational analysis (GRA) and response surface methodology, and is adopted to improve tensile strength and reduce residual stress. In the winding process, the main process parameters involving winding tension, pressure, temperature and speed are selected to evaluate the parametric influences on tensile strength and residual stress. Experiments are conducted using the Box-Behnken design. Based on principal component analysis, the grey relational grades are properly established to convert multi-responses into an individual objective problem. Then the response surface method is used to build a second-order model of grey relational grade and predict the optimum parameters. The predictive accuracy of the developed model is proved by two test experiments with a low prediction error of less than 7%. The following process parameters, namely winding tension 124.29 N, pressure 2000 N, temperature 40 °C and speed 10.65 rpm, have the highest grey relational grade and give better quality characteristics in terms of tensile strength and residual stress. The confirmation experiment shows that better results are obtained with GRA improved by the proposed method than with ordinary GRA. The proposed method is proved to be feasible and can be applied to optimize the multi-objective problem in the filament winding process.

Estimation of glucose rate of appearance from cgs and subcutaneous insulin delivery in type 1 diabetes

KAUST Repository

Laleg-Kirati, Taous-Meriem

2017-08-31

Method and System for providing estimates of Glucose Rate of Appearance from the intestine (GRA) using continuous glucose sensor measurements (CGS) taken from the subcutaneous of a diabetes patient and the amount of insulin administered to the patient.

Discrimination of citrus tristeza virus (CTV) strains using Mexican ...

African Journals Online (AJOL)

Administrator

2007-02-19

Feb 19, 2007 ... little damage to severe decline, especially on trees gra- fted on sour ... The damages and losses caused by ... Stem pitting indicates projections of bark in ... evaluation of stem-pitting reaction level after having removed the.

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

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Emilie Fugier

2009-06-01

Full Text Available The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER-derived replicative organelle named the "Brucella-containing vacuole" (BCV. Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

Pervious Pavement System Evaluation- Abstract 1

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Porous pavement is a low impact development stormwater control. The Urban Watershed Management Branch is evaluating interlocking concrete pavers as a popular implementation. The pavers themselves are impermeable, but the spaces between the pavers are backfilled with washed, gra...

Pervious Pavement System Evaluation- Abstract

Science.gov (United States)

Porous pavement is a low impact development stormwater control. The Urban Watershed Management Branch is evaluating interlocking concrete pavers as a popular implementation. The pavers themselves are impermeable, but the spaces between the pavers are backfilled with washed, gra...

ANALYSIS OF BACTERIAL COMMUNITIES IN SEAGRASS BED SEDIMENTS BY DOUBLE-GRADIENT DENATURING GRADIENT GEL ELECTROPHORESIS OF PCR-AMPLIFIED 16SRRNA GENES

Science.gov (United States)

Bacterial communities associated with seagrass bed sediments are not well studied. The work presented here investigated several factors, including the presence or absence of vegetation, depth into sediment, and season, and their impact on bacterial community diversity. Double gra...

GRA prospectus: optimizing design and management of protected areas

Science.gov (United States)

Bernknopf, Richard; Halsing, David

2001-01-01

Protected areas comprise one major type of global conservation effort that has been in the form of parks, easements, or conservation concessions. Though protected areas are increasing in number and size throughout tropical ecosystems, there is no systematic method for optimally targeting specific local areas for protection, designing the protected area, and monitoring it, or for guiding follow-up actions to manage it or its surroundings over the long run. Without such a system, conservation projects often cost more than necessary and/or risk protecting ecosystems and biodiversity less efficiently than desired. Correcting these failures requires tools and strategies for improving the placement, design, and long-term management of protected areas. The objective of this project is to develop a set of spatially based analytical tools to improve the selection, design, and management of protected areas. In this project, several conservation concessions will be compared using an economic optimization technique. The forest land use portfolio model is an integrated assessment that measures investment in different land uses in a forest. The case studies of individual tropical ecosystems are developed as forest (land) use and preservation portfolios in a geographic information system (GIS). Conservation concessions involve a private organization purchasing development and resource access rights in a certain area and retiring them. Forests are put into conservation, and those people who would otherwise have benefited from extracting resources or selling the right to do so are compensated. Concessions are legal agreements wherein the exact amount and nature of the compensation result from a negotiated agreement between an agent of the conservation community and the local community. Funds are placed in a trust fund, and annual payments are made to local communities and regional/national governments. The payments are made pending third-party verification that the forest expanse and quality have been maintained.

V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

Energy Technology Data Exchange (ETDEWEB)

Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko; Kojima, Ai; Nishinoaki, Show [Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Toshima, Junko Y., E-mail: yama_jun@aoni.waseda.jp [Faculty of Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480 (Japan); Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Toshima, Jiro, E-mail: jtosiscb@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan)

2014-01-10

Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1p in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V{sub o} subunit of vacuolar-type H{sup +}-ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling.

V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

International Nuclear Information System (INIS)

Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko; Kojima, Ai; Nishinoaki, Show; Toshima, Junko Y.; Toshima, Jiro

2014-01-01

Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1p in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V o subunit of vacuolar-type H + -ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling

Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein.

Science.gov (United States)

Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M; Di Guilmi, Anne Marie; Frachet, Philippe

2012-12-14

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.

Human and Pneumococcal Cell Surface Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Proteins Are Both Ligands of Human C1q Protein*

Science.gov (United States)

Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M.; Di Guilmi, Anne Marie; Frachet, Philippe

2012-01-01

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (KD = 0.34–2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response. PMID:23086952

March1 E3 Ubiquitin Ligase Modulates Features of Allergic Asthma in an Ovalbumin-Induced Mouse Model of Lung Inflammation

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Osama A. Kishta

2018-01-01

Full Text Available Membrane-associated RING-CH-1 (March1 is a member of the March family of E3 ubiquitin ligases. March1 downregulates cell surface expression of MHC II and CD86 by targeting them to lysosomal degradation. Given the key roles of MHC class II and CD86 in T cell activation and to get further insights into the development of allergic inflammation, we asked whether March1 deficiency exacerbates or attenuates features of allergic asthma in mice. Herein, we used an acute model of allergy to compare the asthmatic phenotype of March1-deficient and -sufficient mice immunized with ovalbumin (OVA and later challenged by intranasal instillation of OVA in the lungs. We found that eosinophilic inflammation in airways and lung tissue was similar between WT and March1−/− allergic mice, whereas neutrophilic inflammation was significant only in March1−/− mice. Airway hyperresponsiveness as well as levels of IFN-γ, IL-13, IL-6, and IL-10 was lower in the lungs of asthmatic March1−/− mice compared to WT, whereas lung levels of TNF-α, IL-4, and IL-5 were not significantly different. Interestingly, in the serum, levels of total and ova-specific IgE were reduced in March1-deficient mice as compared to WT mice. Taken together, our results demonstrate a role of March1 E3 ubiquitin ligase in modulating allergic responses.

Associations of Physical Activity, Sports Participation and Active Commuting on Mathematic Performance and Inhibitory Control in Adolescents

DEFF Research Database (Denmark)

Domazet, Sidsel L; Tarp, Jakob; Huang, Tao

2016-01-01

OBJECTIVES: To examine objectively measured physical activity level, organized sports participation and active commuting to school in relation to mathematic performance and inhibitory control in adolescents. METHODS: The design was cross-sectional. A convenient sample of 869 sixth and seventh gra...

Effects of red grape skin and seed extract supplementation on atherosclerosis in Watanabe heritable hyperlipidemic rabbits

DEFF Research Database (Denmark)

Frederiksen, Hanne; Mortensen, Alicja; Schrøder, Malene

2007-01-01

Epidemiological studies have suggested an association between consumption of red wine and other polyphenolic compounds and prevention of cardiovascular diseases. In the present study, Watanabe heritable hyperlipidemic (WHHL) rabbits were used to investigate the effects of polyphenols in a red gra...

Unsolicited information letters to increase awareness of Lynch syndrome and familial colorectal cancer

DEFF Research Database (Denmark)

Petersen, Helle Vendel; Frederiksen, Birgitte Lidegaard; Lautrup, Charlotte Kvist

2018-01-01

Dissemination of information on a genetically increased risk should according to guidelines primarily be family-mediated. Incomplete and incorrect information spread has, however, been documented and implies missed possibilities for prevention. In Denmark, the national HNPCC register has been gra...

Durability of lightweight concrete : Phase I : concrete temperature study.

Science.gov (United States)

1968-08-01

This report describes a study conducted to determine the temperature gradient throughout the depth of a six inch concrete bridge deck. The bridge deck selected for study was constructed using lightweight concrete for the center spans and sand and gra...

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

Science.gov (United States)

Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

2016-02-15

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.