WorldWideScience

Sample records for secondary transport proteins

  1. Effect of External Electric Field on Substrate Transport of a Secondary Active Transporter.

    Science.gov (United States)

    Zhang, Ji-Long; Zheng, Qing-Chuan; Yu, Li-Ying; Li, Zheng-Qiang; Zhang, Hong-Xing

    2016-08-22

    Substrate transport across a membrane accomplished by a secondary active transporter (SAT) is essential to the normal physiological function of living cells. In the present research, a series of all-atom molecular dynamics (MD) simulations under different electric field (EF) strengths was performed to investigate the effect of an external EF on the substrate transport of an SAT. The results show that EF both affects the interaction between substrate and related protein's residues by changing their conformations and tunes the timeline of the transport event, which collectively reduces the height of energy barrier for substrate transport and results in the appearance of two intermediate conformations under the existence of an external EF. Our work spotlights the crucial influence of external EFs on the substrate transport of SATs and could provide a more penetrating understanding of the substrate transport mechanism of SATs.

  2. Transport proteins promoting Escherichia coli pathogenesis

    Science.gov (United States)

    Tang, Fengyi; Saier, Milton H.

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. PMID:24747185

  3. Transport proteins promoting Escherichia coli pathogenesis.

    Science.gov (United States)

    Tang, Fengyi; Saier, Milton H

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter

    NARCIS (Netherlands)

    Mulligan, Christopher; Geertsma, Eric R.; Severi, Emmanuele; Kelly, David J.; Poolman, Bert; Thomas, Gavin H.

    2009-01-01

    Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific

  5. Amino acid code of protein secondary structure.

    Science.gov (United States)

    Shestopalov, B V

    2003-01-01

    The calculation of protein three-dimensional structure from the amino acid sequence is a fundamental problem to be solved. This paper presents principles of the code theory of protein secondary structure, and their consequence--the amino acid code of protein secondary structure. The doublet code model of protein secondary structure, developed earlier by the author (Shestopalov, 1990), is part of this theory. The theory basis are: 1) the name secondary structure is assigned to the conformation, stabilized only by the nearest (intraresidual) and middle-range (at a distance no more than that between residues i and i + 5) interactions; 2) the secondary structure consists of regular (alpha-helical and beta-structural) and irregular (coil) segments; 3) the alpha-helices, beta-strands and coil segments are encoded, respectively, by residue pairs (i, i + 4), (i, i + 2), (i, i = 1), according to the numbers of residues per period, 3.6, 2, 1; 4) all such pairs in the amino acid sequence are codons for elementary structural elements, or structurons; 5) the codons are divided into 21 types depending on their strength, i.e. their encoding capability; 6) overlappings of structurons of one and the same structure generate the longer segments of this structure; 7) overlapping of structurons of different structures is forbidden, and therefore selection of codons is required, the codon selection is hierarchic; 8) the code theory of protein secondary structure generates six variants of the amino acid code of protein secondary structure. There are two possible kinds of model construction based on the theory: the physical one using physical properties of amino acid residues, and the statistical one using results of statistical analysis of a great body of structural data. Some evident consequences of the theory are: a) the theory can be used for calculating the secondary structure from the amino acid sequence as a partial solution of the problem of calculation of protein three

  6. Thermodynamic secrets of multidrug resistance: A new take on transport mechanisms of secondary active antiporters.

    Science.gov (United States)

    Zhang, Xuejun C; Liu, Min; Lu, Guangyuan; Heng, Jie

    2018-03-01

    Multidrug resistance (MDR) presents a growing challenge to global public health. Drug extrusion transporters play a critical part in MDR; thus, their mechanisms of substrate recognition are being studied in great detail. In this work, we review common structural features of key transporters involved in MDR. Based on our membrane potential-driving hypothesis, we propose a general energy-coupling mechanism for secondary-active antiporters. This putative mechanism provides a common framework for understanding poly-specificity of most-if not all-MDR transporters. © 2017 The Protein Society.

  7. Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

    Science.gov (United States)

    Vergara-Jaque, Ariela; Fenollar-Ferrer, Cristina; Kaufmann, Desirée; Forrest, Lucy R

    2015-01-01

    Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also

  8. Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms

    Directory of Open Access Journals (Sweden)

    Cristina eFenollar Ferrer

    2015-09-01

    Full Text Available Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to either the outside or inside of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (asymmetry of these systems has been successfully used as a bioinformatic tool, called repeat-swap modeling to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that

  9. Chlorovirus-mediated membrane depolarization of Chlorella alters secondary active transport of solutes.

    Science.gov (United States)

    Agarkova, Irina; Dunigan, David; Gurnon, James; Greiner, Timo; Barres, Julia; Thiel, Gerhard; Van Etten, James L

    2008-12-01

    Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake.

  10. 49 CFR 37.27 - Transportation for elementary and secondary education systems.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Transportation for elementary and secondary education systems. 37.27 Section 37.27 Transportation Office of the Secretary of Transportation... elementary and secondary education systems. (a) The requirements of this part do not apply to public school...

  11. Transporter taxonomy - a comparison of different transport protein classification schemes.

    Science.gov (United States)

    Viereck, Michael; Gaulton, Anna; Digles, Daniela; Ecker, Gerhard F

    2014-06-01

    Currently, there are more than 800 well characterized human membrane transport proteins (including channels and transporters) and there are estimates that about 10% (approx. 2000) of all human genes are related to transport. Membrane transport proteins are of interest as potential drug targets, for drug delivery, and as a cause of side effects and drug–drug interactions. In light of the development of Open PHACTS, which provides an open pharmacological space, we analyzed selected membrane transport protein classification schemes (Transporter Classification Database, ChEMBL, IUPHAR/BPS Guide to Pharmacology, and Gene Ontology) for their ability to serve as a basis for pharmacology driven protein classification. A comparison of these membrane transport protein classification schemes by using a set of clinically relevant transporters as use-case reveals the strengths and weaknesses of the different taxonomy approaches.

  12. Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

    Science.gov (United States)

    Simmons, Katie J; Jackson, Scott M; Brueckner, Florian; Patching, Simon G; Beckstein, Oliver; Ivanova, Ekaterina; Geng, Tian; Weyand, Simone; Drew, David; Lanigan, Joseph; Sharples, David J; Sansom, Mark SP; Iwata, So; Fishwick, Colin WG; Johnson, A Peter; Cameron, Alexander D; Henderson, Peter JF

    2014-01-01

    The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1. PMID:24952894

  13. In Vitro Analysis of Metabolite Transport Proteins.

    Science.gov (United States)

    Roell, Marc-Sven; Kuhnert, Franziska; Zamani-Nour, Shirin; Weber, Andreas P M

    2017-01-01

    The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete. The photorespiratory cycle requires at least 25 transmembrane transport steps; however to date only plastidic glycolate/glycerate transporter and the accessory 2-oxoglutarate/malate and glutamate/malate transporters as well as the mitochondrial transporter BOU1 have been identified. The characterization of transport proteins and defining their substrates and kinetics are still major challenges.Here we present a detailed set of protocols for the in vitro characterization of transport proteins. We provide protocols for the isolation of recombinant transport protein expressed in E. coli or Saccharomyces cerevisiae and the extraction of total leaf membrane protein for in vitro analysis of transporter proteins. Further we explain the process of reconstituting transport proteins in artificial lipid vesicles and elucidate the details of transport assays.

  14. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  15. The Membrane Topology of ALMT1, an Aluminum-Activated Malate Transport Protein in Wheat (Triticum aestivum)

    OpenAIRE

    Motoda, Hirotoshi; Sasaki, Takayuki; Kano, Yoshio; Ryan, Peter R; Delhaize, Emmanuel; Matsumoto, Hideaki; Yamamoto, Yoko

    2007-01-01

    The wheat ALMT1 gene encodes an aluminum (Al)-activated malate transport protein which confers Al-resistance. We investigated the membrane topology of this plasma-membrane localized protein with immunocytochemical techniques. Several green fluorescent protein (GFP)-fused and histidine (His)-tagged chimeras of ALMT1 were prepared based on a computer-predicted secondary structure and transiently expressed in cultured mammalian cells. Antibodies raised to polypeptide epitopes of ALMT1 were used ...

  16. Comparative analyses of transport proteins encoded within the genomes of Leptospira species.

    Science.gov (United States)

    Buyuktimkin, Bora; Saier, Milton H

    2016-09-01

    Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they all have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity arose in Leptospira correlating to progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. Copyright © 2016. Published by Elsevier Ltd.

  17. Comparative genomic analyses of transport proteins encoded within the genomes of Leptospira species.

    Science.gov (United States)

    Buyuktimkin, Bora; Saier, Milton H

    2015-11-01

    Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity was accompanied by progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Proteins mediating intra- and intercellular transport of lipids and lipid-modified proteins

    NARCIS (Netherlands)

    Neumann, S.

    2008-01-01

    Proteins mediating intra- and intercellular transport of lipids and lipid-modified proteins In this thesis, I studied the intra- and intercellular transport of lipidic molecules, in particular glycosphingolipids and lipid-modified proteins. The first part focuses on the intracellular transport of

  19. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

    Science.gov (United States)

    Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

    2014-01-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

  20. Combining neural networks for protein secondary structure prediction

    DEFF Research Database (Denmark)

    Riis, Søren Kamaric

    1995-01-01

    In this paper structured neural networks are applied to the problem of predicting the secondary structure of proteins. A hierarchical approach is used where specialized neural networks are designed for each structural class and then combined using another neural network. The submodels are designed...... by using a priori knowledge of the mapping between protein building blocks and the secondary structure and by using weight sharing. Since none of the individual networks have more than 600 adjustable weights over-fitting is avoided. When ensembles of specialized experts are combined the performance...

  1. Tunneling explains efficient electron transport via protein junctions.

    Science.gov (United States)

    Fereiro, Jerry A; Yu, Xi; Pecht, Israel; Sheves, Mordechai; Cuevas, Juan Carlos; Cahen, David

    2018-05-15

    Metalloproteins, proteins containing a transition metal ion cofactor, are electron transfer agents that perform key functions in cells. Inspired by this fact, electron transport across these proteins has been widely studied in solid-state settings, triggering the interest in examining potential use of proteins as building blocks in bioelectronic devices. Here, we report results of low-temperature (10 K) electron transport measurements via monolayer junctions based on the blue copper protein azurin (Az), which strongly suggest quantum tunneling of electrons as the dominant charge transport mechanism. Specifically, we show that, weakening the protein-electrode coupling by introducing a spacer, one can switch the electron transport from off-resonant to resonant tunneling. This is a consequence of reducing the electrode's perturbation of the Cu(II)-localized electronic state, a pattern that has not been observed before in protein-based junctions. Moreover, we identify vibronic features of the Cu(II) coordination sphere in transport characteristics that show directly the active role of the metal ion in resonance tunneling. Our results illustrate how quantum mechanical effects may dominate electron transport via protein-based junctions.

  2. Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins.

    Science.gov (United States)

    Wacker, Tobias; Garcia-Celma, Juan J; Lewe, Philipp; Andrade, Susana L A

    2014-07-08

    Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.

  3. CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts.

    Science.gov (United States)

    Hafsa, Noor E; Arndt, David; Wishart, David S

    2015-07-01

    The Chemical Shift Index or CSI 3.0 (http://csi3.wishartlab.com) is a web server designed to accurately identify the location of secondary and super-secondary structures in protein chains using only nuclear magnetic resonance (NMR) backbone chemical shifts and their corresponding protein sequence data. Unlike earlier versions of CSI, which only identified three types of secondary structure (helix, β-strand and coil), CSI 3.0 now identifies total of 11 types of secondary and super-secondary structures, including helices, β-strands, coil regions, five common β-turns (type I, II, I', II' and VIII), β hairpins as well as interior and edge β-strands. CSI 3.0 accepts experimental NMR chemical shift data in multiple formats (NMR Star 2.1, NMR Star 3.1 and SHIFTY) and generates colorful CSI plots (bar graphs) and secondary/super-secondary structure assignments. The output can be readily used as constraints for structure determination and refinement or the images may be used for presentations and publications. CSI 3.0 uses a pipeline of several well-tested, previously published programs to identify the secondary and super-secondary structures in protein chains. Comparisons with secondary and super-secondary structure assignments made via standard coordinate analysis programs such as DSSP, STRIDE and VADAR on high-resolution protein structures solved by X-ray and NMR show >90% agreement between those made with CSI 3.0. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Protein secondary structure: category assignment and predictability

    DEFF Research Database (Denmark)

    Andersen, Claus A.; Bohr, Henrik; Brunak, Søren

    2001-01-01

    In the last decade, the prediction of protein secondary structure has been optimized using essentially one and the same assignment scheme known as DSSP. We present here a different scheme, which is more predictable. This scheme predicts directly the hydrogen bonds, which stabilize the secondary......-forward neural network with one hidden layer on a data set identical to the one used in earlier work....

  5. Density functional study of molecular interactions in secondary structures of proteins.

    Science.gov (United States)

    Takano, Yu; Kusaka, Ayumi; Nakamura, Haruki

    2016-01-01

    Proteins play diverse and vital roles in biology, which are dominated by their three-dimensional structures. The three-dimensional structure of a protein determines its functions and chemical properties. Protein secondary structures, including α-helices and β-sheets, are key components of the protein architecture. Molecular interactions, in particular hydrogen bonds, play significant roles in the formation of protein secondary structures. Precise and quantitative estimations of these interactions are required to understand the principles underlying the formation of three-dimensional protein structures. In the present study, we have investigated the molecular interactions in α-helices and β-sheets, using ab initio wave function-based methods, the Hartree-Fock method (HF) and the second-order Møller-Plesset perturbation theory (MP2), density functional theory, and molecular mechanics. The characteristic interactions essential for forming the secondary structures are discussed quantitatively.

  6. Dynamics of protein aggregation and oligomer formation governed by secondary nucleation

    Energy Technology Data Exchange (ETDEWEB)

    Michaels, Thomas C. T., E-mail: tctm3@cam.ac.uk; Lazell, Hamish W.; Arosio, Paolo; Knowles, Tuomas P. J., E-mail: tpjk2@cam.ac.uk [Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW (United Kingdom)

    2015-08-07

    The formation of aggregates in many protein systems can be significantly accelerated by secondary nucleation, a process where existing assemblies catalyse the nucleation of new species. In particular, secondary nucleation has emerged as a central process controlling the proliferation of many filamentous protein structures, including molecular species related to diseases such as sickle cell anemia and a range of neurodegenerative conditions. Increasing evidence suggests that the physical size of protein filaments plays a key role in determining their potential for deleterious interactions with living cells, with smaller aggregates of misfolded proteins, oligomers, being particularly toxic. It is thus crucial to progress towards an understanding of the factors that control the sizes of protein aggregates. However, the influence of secondary nucleation on the time evolution of aggregate size distributions has been challenging to quantify. This difficulty originates in large part from the fact that secondary nucleation couples the dynamics of species distant in size space. Here, we approach this problem by presenting an analytical treatment of the master equation describing the growth kinetics of linear protein structures proliferating through secondary nucleation and provide closed-form expressions for the temporal evolution of the resulting aggregate size distribution. We show how the availability of analytical solutions for the full filament distribution allows us to identify the key physical parameters that control the sizes of growing protein filaments. Furthermore, we use these results to probe the dynamics of the populations of small oligomeric species as they are formed through secondary nucleation and discuss the implications of our work for understanding the factors that promote or curtail the production of these species with a potentially high deleterious biological activity.

  7. Detection of secondary structure elements in proteins by hydrophobic cluster analysis.

    Science.gov (United States)

    Woodcock, S; Mornon, J P; Henrissat, B

    1992-10-01

    Hydrophobic cluster analysis (HCA) is a protein sequence comparison method based on alpha-helical representations of the sequences where the size, shape and orientation of the clusters of hydrophobic residues are primarily compared. The effectiveness of HCA has been suggested to originate from its potential ability to focus on the residues forming the hydrophobic core of globular proteins. We have addressed the robustness of the bidimensional representation used for HCA in its ability to detect the regular secondary structure elements of proteins. Various parameters have been studied such as those governing cluster size and limits, the hydrophobic residues constituting the clusters as well as the potential shift of the cluster positions with respect to the position of the regular secondary structure elements. The following results have been found to support the alpha-helical bidimensional representation used in HCA: (i) there is a positive correlation (clearly above background noise) between the hydrophobic clusters and the regular secondary structure elements in proteins; (ii) the hydrophobic clusters are centred on the regular secondary structure elements; (iii) the pitch of the helical representation which gives the best correspondence is that of an alpha-helix. The correspondence between hydrophobic clusters and regular secondary structure elements suggests a way to implement variable gap penalties during the automatic alignment of protein sequences.

  8. Semantic role labeling for protein transport predicates

    Directory of Open Access Journals (Sweden)

    Martin James H

    2008-06-01

    Full Text Available Abstract Background Automatic semantic role labeling (SRL is a natural language processing (NLP technique that maps sentences to semantic representations. This technique has been widely studied in the recent years, but mostly with data in newswire domains. Here, we report on a SRL model for identifying the semantic roles of biomedical predicates describing protein transport in GeneRIFs – manually curated sentences focusing on gene functions. To avoid the computational cost of syntactic parsing, and because the boundaries of our protein transport roles often did not match up with syntactic phrase boundaries, we approached this problem with a word-chunking paradigm and trained support vector machine classifiers to classify words as being at the beginning, inside or outside of a protein transport role. Results We collected a set of 837 GeneRIFs describing movements of proteins between cellular components, whose predicates were annotated for the semantic roles AGENT, PATIENT, ORIGIN and DESTINATION. We trained these models with the features of previous word-chunking models, features adapted from phrase-chunking models, and features derived from an analysis of our data. Our models were able to label protein transport semantic roles with 87.6% precision and 79.0% recall when using manually annotated protein boundaries, and 87.0% precision and 74.5% recall when using automatically identified ones. Conclusion We successfully adapted the word-chunking classification paradigm to semantic role labeling, applying it to a new domain with predicates completely absent from any previous studies. By combining the traditional word and phrasal role labeling features with biomedical features like protein boundaries and MEDPOST part of speech tags, we were able to address the challenges posed by the new domain data and subsequently build robust models that achieved F-measures as high as 83.1. This system for extracting protein transport information from Gene

  9. PCI-SS: MISO dynamic nonlinear protein secondary structure prediction

    Directory of Open Access Journals (Sweden)

    Aboul-Magd Mohammed O

    2009-07-01

    Full Text Available Abstract Background Since the function of a protein is largely dictated by its three dimensional configuration, determining a protein's structure is of fundamental importance to biology. Here we report on a novel approach to determining the one dimensional secondary structure of proteins (distinguishing α-helices, β-strands, and non-regular structures from primary sequence data which makes use of Parallel Cascade Identification (PCI, a powerful technique from the field of nonlinear system identification. Results Using PSI-BLAST divergent evolutionary profiles as input data, dynamic nonlinear systems are built through a black-box approach to model the process of protein folding. Genetic algorithms (GAs are applied in order to optimize the architectural parameters of the PCI models. The three-state prediction problem is broken down into a combination of three binary sub-problems and protein structure classifiers are built using 2 layers of PCI classifiers. Careful construction of the optimization, training, and test datasets ensures that no homology exists between any training and testing data. A detailed comparison between PCI and 9 contemporary methods is provided over a set of 125 new protein chains guaranteed to be dissimilar to all training data. Unlike other secondary structure prediction methods, here a web service is developed to provide both human- and machine-readable interfaces to PCI-based protein secondary structure prediction. This server, called PCI-SS, is available at http://bioinf.sce.carleton.ca/PCISS. In addition to a dynamic PHP-generated web interface for humans, a Simple Object Access Protocol (SOAP interface is added to permit invocation of the PCI-SS service remotely. This machine-readable interface facilitates incorporation of PCI-SS into multi-faceted systems biology analysis pipelines requiring protein secondary structure information, and greatly simplifies high-throughput analyses. XML is used to represent the input

  10. The molecular mechanism of ion-dependent gating in secondary transporters.

    Directory of Open Access Journals (Sweden)

    Chunfeng Zhao

    2013-10-01

    Full Text Available LeuT-like fold Na-dependent secondary active transporters form a large family of integral membrane proteins that transport various substrates against their concentration gradient across lipid membranes, using the free energy stored in the downhill concentration gradient of sodium ions. These transporters play an active role in synaptic transmission, the delivery of key nutrients, and the maintenance of osmotic pressure inside the cell. It is generally believed that binding of an ion and/or a substrate drives the conformational dynamics of the transporter. However, the exact mechanism for converting ion binding into useful work has yet to be established. Using a multi-dimensional path sampling (string-method followed by all-atom free energy simulations, we established the principal thermodynamic and kinetic components governing the ion-dependent conformational dynamics of a LeuT-like fold transporter, the sodium/benzyl-hydantoin symporter Mhp1, for an entire conformational cycle. We found that inward-facing and outward-facing states of Mhp1 display nearly the same free energies with an ion absent from the Na2 site conserved across the LeuT-like fold transporters. The barrier separating an apo-state from inward-facing or outward-facing states of the transporter is very low, suggesting stochastic gating in the absence of ion/substrate bound. In contrast, the binding of a Na2 ion shifts the free energy stabilizing the outward-facing state and promoting substrate binding. Our results indicate that ion binding to the Na2 site may also play a key role in the intracellular thin gate dynamics modulation by altering its interactions with the transmembrane helix 5 (TM5. The Potential of Mean Force (PMF computations for a substrate entrance displays two energy minima that correspond to the locations of the main binding site S1 and proposed allosteric S2 binding site. However, it was found that substrate's binds to the site S1 ∼5 kcal/mol more favorable

  11. Improving the accuracy of protein secondary structure prediction using structural alignment

    Directory of Open Access Journals (Sweden)

    Gallin Warren J

    2006-06-01

    Full Text Available Abstract Background The accuracy of protein secondary structure prediction has steadily improved over the past 30 years. Now many secondary structure prediction methods routinely achieve an accuracy (Q3 of about 75%. We believe this accuracy could be further improved by including structure (as opposed to sequence database comparisons as part of the prediction process. Indeed, given the large size of the Protein Data Bank (>35,000 sequences, the probability of a newly identified sequence having a structural homologue is actually quite high. Results We have developed a method that performs structure-based sequence alignments as part of the secondary structure prediction process. By mapping the structure of a known homologue (sequence ID >25% onto the query protein's sequence, it is possible to predict at least a portion of that query protein's secondary structure. By integrating this structural alignment approach with conventional (sequence-based secondary structure methods and then combining it with a "jury-of-experts" system to generate a consensus result, it is possible to attain very high prediction accuracy. Using a sequence-unique test set of 1644 proteins from EVA, this new method achieves an average Q3 score of 81.3%. Extensive testing indicates this is approximately 4–5% better than any other method currently available. Assessments using non sequence-unique test sets (typical of those used in proteome annotation or structural genomics indicate that this new method can achieve a Q3 score approaching 88%. Conclusion By using both sequence and structure databases and by exploiting the latest techniques in machine learning it is possible to routinely predict protein secondary structure with an accuracy well above 80%. A program and web server, called PROTEUS, that performs these secondary structure predictions is accessible at http://wishart.biology.ualberta.ca/proteus. For high throughput or batch sequence analyses, the PROTEUS programs

  12. Protein Secondary Structure Prediction Using AutoEncoder Network and Bayes Classifier

    Science.gov (United States)

    Wang, Leilei; Cheng, Jinyong

    2018-03-01

    Protein secondary structure prediction is belong to bioinformatics,and it's important in research area. In this paper, we propose a new prediction way of protein using bayes classifier and autoEncoder network. Our experiments show some algorithms including the construction of the model, the classification of parameters and so on. The data set is a typical CB513 data set for protein. In terms of accuracy, the method is the cross validation based on the 3-fold. Then we can get the Q3 accuracy. Paper results illustrate that the autoencoder network improved the prediction accuracy of protein secondary structure.

  13. Protein Secondary Structures (α-helix and β-sheet) at a Cellular Level and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the α-helix and β-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of β-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution (∼10 μm). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of α-helixes and β-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of α-helixes (from 47.1% to 36.1%: S-FTIR absorption intensity), increased the

  14. Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    Energy Technology Data Exchange (ETDEWEB)

    Yu,P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the {alpha}-helix and {beta}-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of {beta}-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ({approx}10 {mu}m). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of {alpha}-helixes and {beta}-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of {alpha}-helixes (from 47.1% to 36.1%: S

  15. Transport of secondary electrons and reactive species in ion tracks

    Science.gov (United States)

    Surdutovich, Eugene; Solov'yov, Andrey V.

    2015-08-01

    The transport of reactive species brought about by ions traversing tissue-like medium is analysed analytically. Secondary electrons ejected by ions are capable of ionizing other molecules; the transport of these generations of electrons is studied using the random walk approximation until these electrons remain ballistic. Then, the distribution of solvated electrons produced as a result of interaction of low-energy electrons with water molecules is obtained. The radial distribution of energy loss by ions and secondary electrons to the medium yields the initial radial dose distribution, which can be used as initial conditions for the predicted shock waves. The formation, diffusion, and chemical evolution of hydroxyl radicals in liquid water are studied as well. COST Action Nano-IBCT: Nano-scale Processes Behind Ion-Beam Cancer Therapy.

  16. Comparison of all-electric secondary power systems for civil transport

    Science.gov (United States)

    Renz, David D.

    1992-01-01

    Three separate studies have shown operational, weight, and cost advantages for commercial subsonic transport aircraft using an all-electric secondary power system. The first study in 1982 showed that all-electric secondary power systems produced the second largest benefit compared to four other technology upgrades. The second study in 1985 showed a 10 percent weight and fuel savings using an all-electric high frequency (20 kHz) secondary power system. The last study in 1991 showed a 2 percent weight savings using today's technology (400 Hz) in an all-electric secondary power system. This paper will compare the 20 kHz and 400 Hz studies, analyze the 2 to 10 percent difference in weight savings and comment on the common benefits of the all-electric secondary power system.

  17. Monju secondary heat transport system sodium leak

    International Nuclear Information System (INIS)

    Suzuki, Takeo; Hiroi, Hiroshi; Usami, Shin; Iwata, Koji.

    1996-01-01

    On December 8, 1995, the sodium leakage from the secondary heat transport system (SHTS) occurred in the piping room of the reactor auxiliary building in Monju. The secondary sodium leaked through a temperature sensor, due to the breakaway of the tip of the well tube of the sensor installed near the outlet of the intermediate heat exchanger (IHX) in the C loop of SHTS. The reactor core remained cooled and thus, from the viewpoint of radiological hazards, the safety of the reactor was secured. There were no adverse effects for operating personnel or the surrounding environment. The cause of the well tube failure is considered to result from high cycle fatigue due to flow induced vibrations. Delay in draining the sodium from the leaking loop increased the consequential effects from sodium combustion products. (author)

  18. Knowledge base and neural network approach for protein secondary structure prediction.

    Science.gov (United States)

    Patel, Maulika S; Mazumdar, Himanshu S

    2014-11-21

    Protein structure prediction is of great relevance given the abundant genomic and proteomic data generated by the genome sequencing projects. Protein secondary structure prediction is addressed as a sub task in determining the protein tertiary structure and function. In this paper, a novel algorithm, KB-PROSSP-NN, which is a combination of knowledge base and modeling of the exceptions in the knowledge base using neural networks for protein secondary structure prediction (PSSP), is proposed. The knowledge base is derived from a proteomic sequence-structure database and consists of the statistics of association between the 5-residue words and corresponding secondary structure. The predicted results obtained using knowledge base are refined with a Backpropogation neural network algorithm. Neural net models the exceptions of the knowledge base. The Q3 accuracy of 90% and 82% is achieved on the RS126 and CB396 test sets respectively which suggest improvement over existing state of art methods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. EHD proteins: Key conductors of endocytic transport

    Science.gov (United States)

    Naslavsky, Naava; Caplan, Steve

    2010-01-01

    Regulation of endocytic transport is controlled by an elaborate network of proteins. Rab GTP-binding proteins and their effectors have well-defined roles in mediating specific endocytic transport steps, but until recently, less was known about the four mammalian dynamin-like C-terminal Eps15 Homology Domain (EHD) proteins that also regulate endocytic events. In recent years, however, great strides have been made in understanding the structure and function of these unique proteins. Indeed, a growing body of literature addresses EHD protein structure, interactions with binding partners, functions in mammalian cells, and the generation of various new model systems. Accordingly, this is now an opportune time to pause and review the function and mechanisms of action of EHD proteins, and to highlight some of the challenges and future directions for the field. PMID:21067929

  20. Functional analysis of candidate ABC transporter proteins for sitosterol transport

    DEFF Research Database (Denmark)

    Albrecht, C; Elliott, J I; Sardini, A

    2002-01-01

    implicated in lipid movement and expressed in tissues with a role in sterol synthesis and absorption, might also be involved in sitosterol transport. Transport by the multidrug resistance P-glycoprotein (P-gp; Abcb1), the multidrug resistance-associated protein (Mrp1; Abcc1), the breast cancer resistance...

  1. Mechanistic logic underlying the axonal transport of cytosolic proteins

    Science.gov (United States)

    Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

    2011-01-01

    Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

  2. Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

    Science.gov (United States)

    Barz, W P; Walter, P

    1999-04-01

    Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

  3. Comparison of all-electric secondary power systems for civil subsonic transports

    Science.gov (United States)

    Renz, David D.

    1992-01-01

    Three separate studies have shown operational, weight, and cost advantages for commercial subsonic transport aircraft using an all-electric secondary power system. The first study in 1982 showed that all-electric secondary power systems produced the second largest benefit compared to four other technology upgrades. The second study in 1985 showed a 10 percent weight and fuel savings using an all-electric high frequency (20 kHz) secondary power system. The last study in 1991 showed a 2 percent weight savings using today's technology (400 Hz) in an all-electric secondary power system. This paper will compare the 20 kHz and 400 Hz studies, analyze the 2 to 10 percent difference in weight savings and comment on the common benefits of the all-electric secondary power system.

  4. Protein secondary structure appears to be robust under in silico evolution while protein disorder appears not to be.

    KAUST Repository

    Schaefer, Christian

    2010-01-16

    MOTIVATION: The mutation of amino acids often impacts protein function and structure. Mutations without negative effect sustain evolutionary pressure. We study a particular aspect of structural robustness with respect to mutations: regular protein secondary structure and natively unstructured (intrinsically disordered) regions. Is the formation of regular secondary structure an intrinsic feature of amino acid sequences, or is it a feature that is lost upon mutation and is maintained by evolution against the odds? Similarly, is disorder an intrinsic sequence feature or is it difficult to maintain? To tackle these questions, we in silico mutated native protein sequences into random sequence-like ensembles and monitored the change in predicted secondary structure and disorder. RESULTS: We established that by our coarse-grained measures for change, predictions and observations were similar, suggesting that our results were not biased by prediction mistakes. Changes in secondary structure and disorder predictions were linearly proportional to the change in sequence. Surprisingly, neither the content nor the length distribution for the predicted secondary structure changed substantially. Regions with long disorder behaved differently in that significantly fewer such regions were predicted after a few mutation steps. Our findings suggest that the formation of regular secondary structure is an intrinsic feature of random amino acid sequences, while the formation of long-disordered regions is not an intrinsic feature of proteins with disordered regions. Put differently, helices and strands appear to be maintained easily by evolution, whereas maintaining disordered regions appears difficult. Neutral mutations with respect to disorder are therefore very unlikely.

  5. Protein secondary structure appears to be robust under in silico evolution while protein disorder appears not to be.

    KAUST Repository

    Schaefer, Christian; Schlessinger, Avner; Rost, Burkhard

    2010-01-01

    MOTIVATION: The mutation of amino acids often impacts protein function and structure. Mutations without negative effect sustain evolutionary pressure. We study a particular aspect of structural robustness with respect to mutations: regular protein secondary structure and natively unstructured (intrinsically disordered) regions. Is the formation of regular secondary structure an intrinsic feature of amino acid sequences, or is it a feature that is lost upon mutation and is maintained by evolution against the odds? Similarly, is disorder an intrinsic sequence feature or is it difficult to maintain? To tackle these questions, we in silico mutated native protein sequences into random sequence-like ensembles and monitored the change in predicted secondary structure and disorder. RESULTS: We established that by our coarse-grained measures for change, predictions and observations were similar, suggesting that our results were not biased by prediction mistakes. Changes in secondary structure and disorder predictions were linearly proportional to the change in sequence. Surprisingly, neither the content nor the length distribution for the predicted secondary structure changed substantially. Regions with long disorder behaved differently in that significantly fewer such regions were predicted after a few mutation steps. Our findings suggest that the formation of regular secondary structure is an intrinsic feature of random amino acid sequences, while the formation of long-disordered regions is not an intrinsic feature of proteins with disordered regions. Put differently, helices and strands appear to be maintained easily by evolution, whereas maintaining disordered regions appears difficult. Neutral mutations with respect to disorder are therefore very unlikely.

  6. Protein secondary structure prediction using modular reciprocal bidirectional recurrent neural networks.

    Science.gov (United States)

    Babaei, Sepideh; Geranmayeh, Amir; Seyyedsalehi, Seyyed Ali

    2010-12-01

    The supervised learning of recurrent neural networks well-suited for prediction of protein secondary structures from the underlying amino acids sequence is studied. Modular reciprocal recurrent neural networks (MRR-NN) are proposed to model the strong correlations between adjacent secondary structure elements. Besides, a multilayer bidirectional recurrent neural network (MBR-NN) is introduced to capture the long-range intramolecular interactions between amino acids in formation of the secondary structure. The final modular prediction system is devised based on the interactive integration of the MRR-NN and the MBR-NN structures to arbitrarily engage the neighboring effects of the secondary structure types concurrent with memorizing the sequential dependencies of amino acids along the protein chain. The advanced combined network augments the percentage accuracy (Q₃) to 79.36% and boosts the segment overlap (SOV) up to 70.09% when tested on the PSIPRED dataset in three-fold cross-validation. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  7. [Cloning and expression analysis of a zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein encoding gene in Dendrobium officinale].

    Science.gov (United States)

    Zhang, Gang; Li, Yi-Min; Li, Biao; Zhang, Da-Wei; Guo, Shun-Xing

    2015-01-01

    The zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein (ZIP) plays an important role in the growth and development of plant. In this study, a full length cDNA of ZIP encoding gene, designed as DoZIP1 (GenBank accession KJ946203), was identified from Dendrobium officinale using RT-PCR and RACE. Bioinformatics analysis showed that DoZIP1 consisted of a 1,056 bp open reading frame (ORF) encoded a 351-aa protein with a molecular weight of 37.57 kDa and an isoelectric point (pI) of 6.09. The deduced DoZIP1 protein contained the conserved ZIP domain, and its secondary structure was composed of 50.71% alpha helix, 11.11% extended strand, 36.18% random coil, and beta turn 1.99%. DoZIP1 protein exhibited a signal peptide and eight transmembrane domains, presumably locating in cell membrane. The amino acid sequence had high homology with ZIP proteins from Arabidopsis, alfalfa and rice. A phylogenetic tree analysis demonstrated that DoZIP1 was closely related to AtZIP10 and OsZIP3, and they were clustered into one clade. Real time quantitative PCR analysis demonstrated that the transcription level of DoZIP1 in D. officinale roots was the highest (4.19 fold higher than that of stems), followed by that of leaves (1.12 fold). Molecular characters of DoZIP1 will be useful for further functional determination of the gene involving in the growth and development of D. officinale.

  8. Lead corrosion and transport in simulated secondary feedwater

    International Nuclear Information System (INIS)

    McGarvey, G.B.; Ross, K.J.; McDougall, T.E.; Turner, C.W.

    1998-01-01

    The ubiquitous presence of lead at trace levels in secondary feedwater is a concern to all operators of steam generators and has prompted laboratory studies of its interaction with Inconel 600, Inconel 690, Monel 400 and Incoloy 800. Acute exposures of steam generator alloys to high levels of,lead in the laboratory and in the field have accelerated the degradation of these alloys. There is some disagreement over the role of lead when the exposure is to chronic levels. It has been proposed that most of the present degradation of steam generator tubes is due to low levels of lead although few if any failures have been experimentally linked to lead when sub-parts per billion levels are present in the feedwater. One reason for the difficulty in assigning the role of the lead is related to its possible immobilization on the surfaces of corrosion products or iron oxide films in the feedwater system. We have measured lead adsorption profiles on the three principal corrosion products in the secondary feedwater; magnetite, lepidocrocite and hematite. In all cases, essentially complete adsorption of the lead is achieved at pH values less than that of the feedwater (9-10). If lead is maintained in this adsorbed state, it may be more chemically benign than lead that is free to dissolve in the feedwater and subsequently adsorb on steam generator tube surfaces. In this paper, we report on lead adsorption onto simulated corrosion products under simulated feedwater conditions and propose a physical model for the transport and fate of lead under operating conditions. The nature of lead adsorption onto the surfaces of different corrosion products will be discussed. The desorption behaviour of lead from iron oxide surfaces following different treatment conditions will be used to propose a model for tile transport and probable fate of lead in the secondary feedwater system. (author)

  9. Lead corrosion and transport in simulated secondary feedwater

    Energy Technology Data Exchange (ETDEWEB)

    McGarvey, G.B. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada); Ross, K.J.; McDougall, T.E. [Atomic Energy of Canada Limited, Pinawa, Manitoba (Canada); Turner, C.W. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    1998-07-01

    The ubiquitous presence of lead at trace levels in secondary feedwater is a concern to all operators of steam generators and has prompted laboratory studies of its interaction with Inconel 600, Inconel 690, Monel 400 and Incoloy 800. Acute exposures of steam generator alloys to high levels of,lead in the laboratory and in the field have accelerated the degradation of these alloys. There is some disagreement over the role of lead when the exposure is to chronic levels. It has been proposed that most of the present degradation of steam generator tubes is due to low levels of lead although few if any failures have been experimentally linked to lead when sub-parts per billion levels are present in the feedwater. One reason for the difficulty in assigning the role of the lead is related to its possible immobilization on the surfaces of corrosion products or iron oxide films in the feedwater system. We have measured lead adsorption profiles on the three principal corrosion products in the secondary feedwater; magnetite, lepidocrocite and hematite. In all cases, essentially complete adsorption of the lead is achieved at pH values less than that of the feedwater (9-10). If lead is maintained in this adsorbed state, it may be more chemically benign than lead that is free to dissolve in the feedwater and subsequently adsorb on steam generator tube surfaces. In this paper, we report on lead adsorption onto simulated corrosion products under simulated feedwater conditions and propose a physical model for the transport and fate of lead under operating conditions. The nature of lead adsorption onto the surfaces of different corrosion products will be discussed. The desorption behaviour of lead from iron oxide surfaces following different treatment conditions will be used to propose a model for tile transport and probable fate of lead in the secondary feedwater system. (author)

  10. Bayesian Inference using Neural Net Likelihood Models for Protein Secondary Structure Prediction

    Directory of Open Access Journals (Sweden)

    Seong-Gon Kim

    2011-06-01

    Full Text Available Several techniques such as Neural Networks, Genetic Algorithms, Decision Trees and other statistical or heuristic methods have been used to approach the complex non-linear task of predicting Alpha-helicies, Beta-sheets and Turns of a proteins secondary structure in the past. This project introduces a new machine learning method by using an offline trained Multilayered Perceptrons (MLP as the likelihood models within a Bayesian Inference framework to predict secondary structures proteins. Varying window sizes are used to extract neighboring amino acid information and passed back and forth between the Neural Net models and the Bayesian Inference process until there is a convergence of the posterior secondary structure probability.

  11. Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD

    Directory of Open Access Journals (Sweden)

    Moon Yuseok

    2009-01-01

    Full Text Available Abstract Background ATP binding cassette (ABC transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA, lipase ABC transporter domains (LARDs were designed for the secretion of fusion proteins. Results The LARDs included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD to three types of proteins; green fluorescent protein (GFP, epidermal growth factor (EGF and cytoplasmic transduction peptide (CTP. These fusion proteins were expressed in Escherichia coli together with ABC transporter of either P. fluorescens or Erwinia chrysanthemi. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant. Conclusion The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in E. coli supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in Pseudomonas using LARDs or TliA as a C-terminal signal sequence.

  12. Water Transport Mediated by Other Membrane Proteins.

    Science.gov (United States)

    Huang, Boyue; Wang, Hongkai; Yang, Baoxue

    2017-01-01

    Water transport through membrane is so intricate that there are still some debates. (Aquaporins) AQPs are entirely accepted to allow water transmembrane movement depending on osmotic gradient. Cotransporters and uniporters , however, are also concerned in water homeotatsis. Urea transporter B (UT-B) has a single-channel water permeability that is similar to AQP1. Cystic fibrosis transmembrane conductance regulator (CFTR ) was initially thought as a water channel but now not believed to transport water directly. By cotranporters, water is transported by water osmosis coupling with substrates, which explains how water is transported across the isolated small intestine. This chapter provides information about water transport mediated by other membrane proteins except AQPs .

  13. Irradiation effects on secondary structure of protein induced by keV ions

    International Nuclear Information System (INIS)

    Cui, F.Z.; Lin, Y.B.; Zhang, D.M.; Tian, M.B.

    2001-01-01

    Protein secondary structure changes by low-energy ion irradiation are reported for the first time. The selected system is 30 keV N + irradiation on bovine serum albumin (BSA). After irradiation at increasing fluences from 1.0x10 15 to 2.5x10 16 ion/cm 2 , Fourier transform infrared spectra analysis was conducted. It was found that the secondary structures of BSA molecules were very sensitive to ion irradiation. Secondary conformations showed different trends of change during irradiation. With the increase of ion fluence from 0 to 2.5x10 16 ion/cm 2 , the fraction of α-helix and β-turns decreased from 17 to 12%, and from 40 to 31%, respectively, while that of random coil and β-sheet structure increased from 18 to 27%, and from 25 to 30%, respectively. Possible explanations for the secondary conformational changes of protein are proposed. (author)

  14. A Kernel for Protein Secondary Structure Prediction

    OpenAIRE

    Guermeur , Yann; Lifchitz , Alain; Vert , Régis

    2004-01-01

    http://mitpress.mit.edu/catalog/item/default.asp?ttype=2&tid=10338&mode=toc; International audience; Multi-class support vector machines have already proved efficient in protein secondary structure prediction as ensemble methods, to combine the outputs of sets of classifiers based on different principles. In this chapter, their implementation as basic prediction methods, processing the primary structure or the profile of multiple alignments, is investigated. A kernel devoted to the task is in...

  15. MUFOLD-SS: New deep inception-inside-inception networks for protein secondary structure prediction.

    Science.gov (United States)

    Fang, Chao; Shang, Yi; Xu, Dong

    2018-05-01

    Protein secondary structure prediction can provide important information for protein 3D structure prediction and protein functions. Deep learning offers a new opportunity to significantly improve prediction accuracy. In this article, a new deep neural network architecture, named the Deep inception-inside-inception (Deep3I) network, is proposed for protein secondary structure prediction and implemented as a software tool MUFOLD-SS. The input to MUFOLD-SS is a carefully designed feature matrix corresponding to the primary amino acid sequence of a protein, which consists of a rich set of information derived from individual amino acid, as well as the context of the protein sequence. Specifically, the feature matrix is a composition of physio-chemical properties of amino acids, PSI-BLAST profile, and HHBlits profile. MUFOLD-SS is composed of a sequence of nested inception modules and maps the input matrix to either eight states or three states of secondary structures. The architecture of MUFOLD-SS enables effective processing of local and global interactions between amino acids in making accurate prediction. In extensive experiments on multiple datasets, MUFOLD-SS outperformed the best existing methods and other deep neural networks significantly. MUFold-SS can be downloaded from http://dslsrv8.cs.missouri.edu/~cf797/MUFoldSS/download.html. © 2018 Wiley Periodicals, Inc.

  16. Fast axonal transport of labeled proteins in motoneurons of exercise-trained rats

    International Nuclear Information System (INIS)

    Jasmin, B.J.; Lavoie, P.A.; Gardiner, P.F.

    1988-01-01

    In this study, the fast orthograde axonal transport of radiolabeled proteins was measured to determine the effects of endurance-running training on transport velocity and amounts of transported proteins in rat sciatic motoneurons. Female rats were subjected to a progressive running-training program for 10-12 wk. Twenty-four hours after the last training session, rats underwent right L4-L5 dorsal root ganglionectomy. The next day, 20 microCi of [3H]leucine was injected bilaterally in the vicinity of the motoneuronal cell bodies supplying the sciatic nerve, to study axonal transport parameters. Results showed that peak and average transport velocities of labeled proteins were significantly (P less than 0.05) increased by 22 and 29%, respectively, in the deafferented nerves of the runners as compared with controls. Moreover, the amount of total transported protein-bound radioactivity was increased in both left (40%) and right (37%) sciatic nerves of the runners. An exhaustive exercise session reduced (P less than 0.05) peak displacement (8%) and total transported protein-bound radioactivity (36%) in the sciatic nerves of control rats, whereas no changes were noticed in trained animals. The data suggest that chronic endurance running induces significant adaptations in the fast axonal transport of labeled proteins

  17. Artificial Intelligence in Prediction of Secondary Protein Structure Using CB513 Database

    Science.gov (United States)

    Avdagic, Zikrija; Purisevic, Elvir; Omanovic, Samir; Coralic, Zlatan

    2009-01-01

    In this paper we describe CB513 a non-redundant dataset, suitable for development of algorithms for prediction of secondary protein structure. A program was made in Borland Delphi for transforming data from our dataset to make it suitable for learning of neural network for prediction of secondary protein structure implemented in MATLAB Neural-Network Toolbox. Learning (training and testing) of neural network is researched with different sizes of windows, different number of neurons in the hidden layer and different number of training epochs, while using dataset CB513. PMID:21347158

  18. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    Science.gov (United States)

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

  19. Tchebichef image moment approach to the prediction of protein secondary structures based on circular dichroism.

    Science.gov (United States)

    Li, Sha Sha; Li, Bao Qiong; Liu, Jin Jin; Lu, Shao Hua; Zhai, Hong Lin

    2018-04-20

    Circular dichroism (CD) spectroscopy is a widely used technique for the evaluation of protein secondary structures that has a significant impact for the understanding of molecular biology. However, the quantitative analysis of protein secondary structures based on CD spectra is still a hard work due to the serious overlap of the spectra corresponding to different structural motifs. Here, Tchebichef image moment (TM) approach is introduced for the first time, which can effectively extract the chemical features in CD spectra for the quantitative analysis of protein secondary structures. The proposed approach was applied to analyze reference set. and the obtained results were evaluated by the strict statistical parameters such as correlation coefficient, cross-validation correlation coefficient and root mean squared error. Compared with several specialized prediction methods, TM approach provided satisfactory results, especially for turns and unordered structures. Our study indicates that TM approach can be regarded as a feasible tool for the analysis of the secondary structures of proteins based on CD spectra. An available TMs package is provided and can be used directly for secondary structures prediction. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

  20. Quantitative proteome and phosphoproteome analyses of Streptomyces coelicolor reveal proteins and phosphoproteins modulating differentiation and secondary metabolism

    DEFF Research Database (Denmark)

    Rioseras, Beatriz; Sliaha, Pavel V; Gorshkov, Vladimir

    2018-01-01

    identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor proteome across three developmental stages: vegetative hypha (MI); secondary metabolite producing hyphae (MII); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during....../Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of Streptomyces coelicolor. We...... the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signalling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism...

  1. Lead corrosion and transport in simulated secondary feedwater

    Energy Technology Data Exchange (ETDEWEB)

    McGarvey, G.B. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada); Ross, K.J.; McDougall, T.E. [Atomic Energy of Canada Limited, Pinawa, Manitoba (Canada); Turner, C.W

    1999-07-01

    The ubiquitous presence of lead at trace levels in secondary feedwater is a concern to all operators of steam generators and has prompted laboratory studies of its interaction with Inconel 600, Inconel 690, Monel 400 and Incoloy 800. Acute exposures of steam generator alloys to high levels of lead in the laboratory and in the field have accelerated the degradation of these alloys. There is some disagreement over the role of lead when the exposure is to chronic levels. It has been proposed that most of the present degradation of steam generator tubes is caused by low levels of lead although few, if any, failures have been experimentally linked to lead when it is present in sub-parts per billion in the feedwater. One reason for the difficulty in assigning the role of the lead is related to its possible immobilization on the surfaces of corrosion products or iron oxide films in the feedwater system. We have measured lead adsorption profiles on the 3 principal corrosion products in the secondary feedwater: magnetite, lepidocrocite and hematite. In all cases, essentially complete adsorption of the lead is achieved at pH values that are lower than the pH of the feedwater (9 to 10). If lead is maintained in this adsorbed state, it may be more chemically benign than lead that is free to dissolve in the feedwater and subsequently adsorb on steam generator tube surfaces. In this paper, we report on lead adsorption onto simulated corrosion products under simulated feedwater conditions and propose a physical model for the transport and fate of lead under operating conditions. The nature of lead adsorption onto the surfaces of different corrosion products will be discussed. The desorption behaviour of lead from iron oxide surfaces after different treatment conditions will be used to propose a model for the transport and probable fate of lead in the secondary feedwater system. (author)

  2. An Expanding Role For Purine Uptake Permease (PUP -like Transporters In Plant Secondary Metabolism.

    Directory of Open Access Journals (Sweden)

    John G. Jelesko

    2012-05-01

    Full Text Available For the past decade, our understanding of the plant purine uptake permease (PUP transporter family of was primarily oriented on purine nucleobase substrates and their tissue-specific expression patterns in Arabidopsis. However, a tobacco PUP-like homolog demonstrating nicotine uptake permease (NUP activity was recently shown to affect both nicotine metabolism and root cell growth. These new findings expand the physiological role for PUP-like transporters to include plant secondary metabolism. Molecular evolution analyses of PUP-like transporters indicate they are distinct group within an ancient super family of drug and metabolite transporters (DMTs. The PUP-like family originated during terrestrial plant evolution sometime between the bryophytes and the lycophytes. A phylogenetic analysis indicates that the PUP-like transporters were likely were derived from a pre-existing nucleotide sugar transporter family within the DMT super family. Within the lycophyte Selaginella, there are three paralogous groups of PUP-like transporters. One of the three PUP-like paralogous groups showed an extensive pattern of gene duplication and diversification within the angiosperm lineage, whereas the other two more ancestral PUP-like paralogous groups did not. Biochemical characterization of four closely-related PUP-like paralogs together with model-based phylogenetic analyses indicate both subfunctionalization and neofunctionalization during the molecular evolution of angiosperm PUP-like transporters. These findings suggest that members of the PUP-like family of DMT transporters are likely involved in diverse primary and secondary plant metabolic pathways.

  3. Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle

    Science.gov (United States)

    Feng, Liang; Campbell, Ernest B.; Hsiung, Yichun; MacKinnon, Roderick

    2011-01-01

    CLC proteins transport Cl− ions across cell membranes to control the electrical potential of muscle cells, transfer electrolytes across epithelia, and control the pH and electrolyte composition of intracellular organelles. Some members of this protein family are Cl− ion channels, while others are secondary active transporters that exchange Cl− ions and H+ with a 2:1 stoichiometry. We have determined the structure of a eukaryotic CLC transporter at 3.5 Å resolution. Cytoplasmic CBS domains are strategically positioned to regulate the ion transport pathway, and many disease-causing mutations in human CLCs reside on the CBS-transmembrane interface. Comparison with prokaryotic CLC shows that a gating glutamate changes conformation and suggests a basis for 2:1 Cl−/H+ exchange and a simple mechanistic connection between CLC channels and transporters. PMID:20929736

  4. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    Bagaria, A.; Swaminathan, S.; Kumaran, D.; Burley, S. K.

    2011-04-01

    The ATP-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and non-transport related processes such as translation of RNA and DNA repair. Typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP) and Ribose binding protein (RBP). Each of these proteins consists of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations have been reported and so for MBP. The closed/active form of the protein interacts with the integral membrane component of the system in both transport and chemotaxis. Herein, we report 1.9{angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound to

  5. Identifying secondary structures in proteins using NMR chemical shift 3D correlation maps

    Science.gov (United States)

    Kumari, Amrita; Dorai, Kavita

    2013-06-01

    NMR chemical shifts are accurate indicators of molecular environment and have been extensively used as aids in protein structure determination. This work focuses on creating empirical 3D correlation maps of backbone chemical shift nuclei for use as identifiers of secondary structure elements in proteins. A correlated database of backbone nuclei chemical shifts was constructed from experimental structural data gathered from entries in the Protein Data Bank (PDB) as well as isotropic chemical shift values from the RefDB database. Rigorous statistical analysis of the maps led to the conclusion that specific correlations between triplets of backbone chemical shifts are best able to differentiate between different secondary structures such as α-helices, β-strands and turns. The method is compared with similar techniques that use NMR chemical shift information as aids in biomolecular structure determination and performs well in tests done on experimental data determined for different types of proteins, including large multi-domain proteins and membrane proteins.

  6. Protein Recovery from Secondary Paper Sludge and Its Potential Use as Wood Adhesive

    Science.gov (United States)

    Pervaiz, Muhammad

    Secondary sludge is an essential part of biosolids produced through the waste treatment plant of paper mills. Globally paper mills generate around 3.0 million ton of biosolids and in the absence of beneficial applications, the handling and disposal of this residual biomass poses a serious environmental and economic proposition. Secondary paper sludges were investigated in this work for recovery of proteins and their use as wood adhesive. After identifying extracellular polymeric substances as adhesion pre-cursors through analytical techniques, studies were carried out to optimize protein recovery from SS and its comprehensive characterization. A modified physicochemical protocol was developed to recover protein from secondary sludge in substantial quantities. The combined effect of French press and sonication techniques followed by alkali treatment resulted in significant improvement of 44% in the yield of solubilized protein compared to chemical methods. The characterization studies confirmed the presence of common amino acids in recovered sludge protein in significant quantities and heavy metal concentration was reduced after recovery process. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of both low and high molecular weight protein fractions in recovered sludge protein. After establishing the proof-of-concept in the use of recovered sludge protein as wood adhesive, the bonding mechanism of protein adhesives with cellulose substrate was further elucidated in a complementary protein-modification study involving soy protein isolate and its glycinin fractions. The results of this study validated the prevailing bonding theories by proving that surface wetting, protein structure, and type of wood play important role in determining final adhesive strength. Recovered sludge protein was also investigated for its compatibility to formulate hybrid adhesive blends with formaldehyde and bio-based polymers. Apart from chemical

  7. Predicting Protein Secondary Structure with Markov Models

    DEFF Research Database (Denmark)

    Fischer, Paul; Larsen, Simon; Thomsen, Claus

    2004-01-01

    we are considering here, is to predict the secondary structure from the primary one. To this end we train a Markov model on training data and then use it to classify parts of unknown protein sequences as sheets, helices or coils. We show how to exploit the directional information contained...... in the Markov model for this task. Classifications that are purely based on statistical models might not always be biologically meaningful. We present combinatorial methods to incorporate biological background knowledge to enhance the prediction performance....

  8. Training-induced changes in membrane transport proteins of human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, C.

    2006-01-01

    Training improves human physical performance by inducing structural and cardiovascular changes, metabolic changes, and changes in the density of membrane transport proteins. This review focuses on the training-induced changes in proteins involved in sarcolemmal membrane transport. It is concluded...

  9. Analysis of Nanobody-Epitope Interactions in Living Cells via Quantitative Protein Transport Assays.

    Science.gov (United States)

    Früholz, Simone; Pimpl, Peter

    2017-01-01

    Over the past few decades, quantitative protein transport analyses have been used to elucidate the sorting and transport of proteins in the endomembrane system of plants. Here, we have applied our knowledge about transport routes and the corresponding sorting signals to establish an in vivo system for testing specific interactions between soluble proteins.Here, we describe the use of quantitative protein transport assays in tobacco mesophyll protoplasts to test for interactions occurring between a GFP-binding nanobody and its GFP epitope. For this, we use a secreted GFP-tagged α-amylase as a reporter together with a vacuolar-targeted RFP-tagged nanobody. The interaction between these proteins is then revealed by a transport alteration of the secretory reporter due to the interaction-triggered attachment of the vacuolar sorting signal.

  10. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    Science.gov (United States)

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-04

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

  11. Myelin-associated proteins labelled by slow axonal transport

    International Nuclear Information System (INIS)

    Giorgi, P.P.; DuBois, H.

    1981-01-01

    This paper deals with the problem of protein metabolism and provides evidence that the neuronal contribution to myelin metabolism may be restricted to lipids only. On the other hand this line of research led to the partial characterization of a group of neuronal proteins probably involved in axo-glial interactions subserving the onset of myelination and the structural maintenance of the mature myelin sheath. Intraocular injection of radioactive amino acids allows the study of the anterograde transport of labelled proteins along retinofugal fibres which are well myelinated. Myelin extracted from the optic nerve and tract under these conditions also contains labelled proteins. Three hypotheses are available to explain this phenomenon. To offer an explanation for this phenomenon the work was planned as follows. a) Characterization of the spatio-temporal pattern of labelling of myelin, in order to define the experimental conditions (survival time and region of the optic pathway to be studied) necessary to obtain maximal labelling. b) Characterization (by gel electrophoresis) of the myelin-associated proteins which become labelled by axonal transport, in order to work on a consistent pattern of labelling. c) Investigation of the possible mechanism responsible for the labelling of myelin-associated proteins. (Auth.)

  12. The lactococcal secondary multidrug transporter LmrP confers resistance to lincosamides, macrolides, streptogramins and tetracyclines

    NARCIS (Netherlands)

    Putman, M; van Veen, HW; Degener, JE; Konings, WN

    2001-01-01

    The active efflux of toxic compounds by (multi)drug transporters is one of the mechanisms that bacteria have developed to resist cytotoxic drugs. The authors describe the role of the lactococcal secondary multidrug transporter LmrP in the resistance to a broad range of clinically important

  13. Cascaded bidirectional recurrent neural networks for protein secondary structure prediction.

    Science.gov (United States)

    Chen, Jinmiao; Chaudhari, Narendra

    2007-01-01

    Protein secondary structure (PSS) prediction is an important topic in bioinformatics. Our study on a large set of non-homologous proteins shows that long-range interactions commonly exist and negatively affect PSS prediction. Besides, we also reveal strong correlations between secondary structure (SS) elements. In order to take into account the long-range interactions and SS-SS correlations, we propose a novel prediction system based on cascaded bidirectional recurrent neural network (BRNN). We compare the cascaded BRNN against another two BRNN architectures, namely the original BRNN architecture used for speech recognition as well as Pollastri's BRNN that was proposed for PSS prediction. Our cascaded BRNN achieves an overall three state accuracy Q3 of 74.38\\%, and reaches a high Segment OVerlap (SOV) of 66.0455. It outperforms the original BRNN and Pollastri's BRNN in both Q3 and SOV. Specifically, it improves the SOV score by 4-6%.

  14. Structure of a Eukaryotic CLC Transporter Defines an Intermediate State in the Transport Cycle

    International Nuclear Information System (INIS)

    Feng, Liang; Campbell, Ernest B.; Hsiung, Yichun; MacKinnon, Roderick

    2010-01-01

    CLC proteins transport chloride (Cl - ) ions across cell membranes to control the electrical potential of muscle cells, transfer electrolytes across epithelia, and control the pH and electrolyte composition of intracellular organelles. Some members of this protein family are Cl - ion channels, whereas others are secondary active transporters that exchange Cl - ions and protons (H + ) with a 2:1 stoichiometry. We have determined the structure of a eukaryotic CLC transporter at 3.5 angstrom resolution. Cytoplasmic cystathionine beta-synthase (CBS) domains are strategically positioned to regulate the ion-transport pathway, and many disease-causing mutations in human CLCs reside on the CBS-transmembrane interface. Comparison with prokaryotic CLC shows that a gating glutamate residue changes conformation and suggests a basis for 2:1 Cl - /H + exchange and a simple mechanistic connection between CLC channels and transporters.

  15. Complement Activation by Ceramide Transporter Proteins

    NARCIS (Netherlands)

    Bode, G.H.; Losen, M.; Buurman, W.A.; Veerhuis, R.; Molenaar, P.C.; Steinbusch, H.W.M.; De Baets, M.H.; Daha, MR; Martinez-Martinez, P.

    2014-01-01

    C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with

  16. Prediction of membrane transport proteins and their substrate specificities using primary sequence information.

    Directory of Open Access Journals (Sweden)

    Nitish K Mishra

    Full Text Available Membrane transport proteins (transporters move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task.Support vector machine (SVM-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM, were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC of 0.49 and a receiver operating characteristic area under the curve (AUC of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset.Our analyses suggest that evolutionary information (i.e., the PSSM and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models do not provide accurate predictions

  17. Parallel protein secondary structure prediction based on neural networks.

    Science.gov (United States)

    Zhong, Wei; Altun, Gulsah; Tian, Xinmin; Harrison, Robert; Tai, Phang C; Pan, Yi

    2004-01-01

    Protein secondary structure prediction has a fundamental influence on today's bioinformatics research. In this work, binary and tertiary classifiers of protein secondary structure prediction are implemented on Denoeux belief neural network (DBNN) architecture. Hydrophobicity matrix, orthogonal matrix, BLOSUM62 and PSSM (position specific scoring matrix) are experimented separately as the encoding schemes for DBNN. The experimental results contribute to the design of new encoding schemes. New binary classifier for Helix versus not Helix ( approximately H) for DBNN produces prediction accuracy of 87% when PSSM is used for the input profile. The performance of DBNN binary classifier is comparable to other best prediction methods. The good test results for binary classifiers open a new approach for protein structure prediction with neural networks. Due to the time consuming task of training the neural networks, Pthread and OpenMP are employed to parallelize DBNN in the hyperthreading enabled Intel architecture. Speedup for 16 Pthreads is 4.9 and speedup for 16 OpenMP threads is 4 in the 4 processors shared memory architecture. Both speedup performance of OpenMP and Pthread is superior to that of other research. With the new parallel training algorithm, thousands of amino acids can be processed in reasonable amount of time. Our research also shows that hyperthreading technology for Intel architecture is efficient for parallel biological algorithms.

  18. Transport proteins of parasitic protists and their role in nutrient salvage.

    Science.gov (United States)

    Dean, Paul; Major, Peter; Nakjang, Sirintra; Hirt, Robert P; Embley, T Martin

    2014-01-01

    The loss of key biosynthetic pathways is a common feature of important parasitic protists, making them heavily dependent on scavenging nutrients from their hosts. This is often mediated by specialized transporter proteins that ensure the nutritional requirements of the parasite are met. Over the past decade, the completion of several parasite genome projects has facilitated the identification of parasite transporter proteins. This has been complemented by functional characterization of individual transporters along with investigations into their importance for parasite survival. In this review, we summarize the current knowledge on transporters from parasitic protists and highlight commonalities and differences in the transporter repertoires of different parasitic species, with particular focus on characterized transporters that act at the host-pathogen interface.

  19. Alterations in protein transport events in rat liver after estrogen treatment

    International Nuclear Information System (INIS)

    Goldsmith, M.A.; Jones, A.L.; Underdown, B.J.; Schiff, J.M.

    1987-01-01

    The effects of 17α-ethynylestradiol (EE) treatment on the hepatic processing of rat polymeric immunoglobulin A (IgA) and human asialoorosomucoid (ASOr) were studied. After 5 days of treatment with EE (5 mg/kg) or solvent alone, male rats were anesthetized and injected with tracer doses of the test proteins. Bile flow rates had been reduced by >60% in the EE-treated animals. A previously reported radiolabeling strategy was used to monitor both the transport of intact protein to bile and the degradation of protein in lysosomes. Transport of intact IgA to bile was reduced by 43%, with transport peaking 27 min later in EE-treated animals compared with controls. There was a corresponding impairment of uptake of labeled IgA from blood. EE induced no kinetic change in the uptake or processing of ASOr. However, there was an increase in the proportion of ASOr reaching bile intact from 3% to 15-23% of the injected dose. The data indicate that EE disables the transport pathway for IgA and causes a partial change in the routing of ASOr after endocytosis in favor of direct transport to the bile canaliculus. These findings may have implications for the importance of membrane composition in protein transport events

  20. New capabilities for Monte Carlo simulation of deuteron transport and secondary products generation

    International Nuclear Information System (INIS)

    Sauvan, P.; Sanz, J.; Ogando, F.

    2010-01-01

    Several important research programs are dedicated to the development of facilities based on deuteron accelerators. In designing these facilities, the definition of a validated computational approach able to simulate deuteron transport and evaluate deuteron interactions and production of secondary particles with acceptable precision is a very important issue. Current Monte Carlo codes, such as MCNPX or PHITS, when applied for deuteron transport calculations use built-in semi-analytical models to describe deuteron interactions. These models are found unreliable in predicting neutron and photon generated by low energy deuterons, typically present in those facilities. We present a new computational tool, resulting from an extension of the MCNPX code, which improve significantly the treatment of problems where any secondary product (neutrons, photons, tritons, etc.) generated by low energy deuterons reactions could play a major role. Firstly, it handles deuteron evaluated data libraries, which allow describing better low deuteron energy interactions. Secondly, it includes a reduction variance technique for production of secondary particles by charged particle-induced nuclear interactions, which allow reducing drastically the computing time needed in transport and nuclear response calculations. Verification of the computational tool is successfully achieved. This tool can be very helpful in addressing design issues such as selection of the dedicated neutron production target and accelerator radioprotection analysis. It can be also helpful to test the deuteron cross-sections under development in the frame of different international nuclear data programs.

  1. Prediction of backbone dihedral angles and protein secondary structure using support vector machines

    Directory of Open Access Journals (Sweden)

    Hirst Jonathan D

    2009-12-01

    Full Text Available Abstract Background The prediction of the secondary structure of a protein is a critical step in the prediction of its tertiary structure and, potentially, its function. Moreover, the backbone dihedral angles, highly correlated with secondary structures, provide crucial information about the local three-dimensional structure. Results We predict independently both the secondary structure and the backbone dihedral angles and combine the results in a loop to enhance each prediction reciprocally. Support vector machines, a state-of-the-art supervised classification technique, achieve secondary structure predictive accuracy of 80% on a non-redundant set of 513 proteins, significantly higher than other methods on the same dataset. The dihedral angle space is divided into a number of regions using two unsupervised clustering techniques in order to predict the region in which a new residue belongs. The performance of our method is comparable to, and in some cases more accurate than, other multi-class dihedral prediction methods. Conclusions We have created an accurate predictor of backbone dihedral angles and secondary structure. Our method, called DISSPred, is available online at http://comp.chem.nottingham.ac.uk/disspred/.

  2. Resveratrol Inhibits Porcine Intestinal Glucose and Alanine Transport: Potential Roles of Na+/K+-ATPase Activity, Protein Kinase A, AMP-Activated Protein Kinase and the Association of Selected Nutrient Transport Proteins with Detergent Resistant Membranes

    Directory of Open Access Journals (Sweden)

    Stefanie Klinger

    2018-03-01

    Full Text Available Background: Beneficial effects of Resveratrol (RSV have been demonstrated, including effects on transporters and channels. However, little is known about how RSV influences intestinal transport. The aim of this study was to further characterize the effects of RSV on intestinal transport and the respective mechanisms. Methods: Porcine jejunum and ileum were incubated with RSV (300 µM, 30 min in Ussing chambers (functional studies and tissue bathes (detection of protein expression, phosphorylation, association with detergent resistant membranes (DRMs. Results: RSV reduced alanine and glucose-induced short circuit currents (ΔIsc and influenced forskolin-induced ΔIsc. The phosphorylation of sodium–glucose-linked transporter 1 (SGLT1, AMP-activated protein kinase (AMPK, protein kinase A substrates (PKA-S and liver kinase B1 (LKB1 increased but a causative relation to the inhibitory effects could not directly be established. The DRM association of SGLT1, peptide transporter 1 (PEPT1 and (phosphorylated Na+/H+-exchanger 3 (NHE3 did not change. Conclusion: RSV influences the intestinal transport of glucose, alanine and chloride and is likely to affect other transport processes. As the effects of protein kinase activation vary between the intestinal localizations, it would appear that increasing cyclic adenosine monophosphate (cAMP levels are part of the mechanism. Nonetheless, the physiological responses depend on cell type-specific structures.

  3. The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lin

    Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

  4. Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase.

    Directory of Open Access Journals (Sweden)

    Kara R Barber

    2017-02-01

    Full Text Available Disruption of synapses underlies a plethora of neurodevelopmental and neurodegenerative disease. Presynaptic specialization called the active zone plays a critical role in the communication with postsynaptic neuron. While the role of many proteins at the active zones in synaptic communication is relatively well studied, very little is known about how these proteins are transported to the synapses. For example, are there distinct mechanisms for the transport of active zone components or are they all transported in the same transport vesicle? Is active zone protein transport regulated? In this report we show that overexpression of Par-1/MARK kinase, a protein whose misregulation has been implicated in Autism spectrum disorders (ASDs and neurodegenerative disorders, lead to a specific block in the transport of an active zone protein component- Bruchpilot at Drosophila neuromuscular junctions. Consistent with a block in axonal transport, we find a decrease in number of active zones and reduced neurotransmission in flies overexpressing Par-1 kinase. Interestingly, we find that Par-1 acts independently of Tau-one of the most well studied substrates of Par-1, revealing a presynaptic function for Par-1 that is independent of Tau. Thus, our study strongly suggests that there are distinct mechanisms that transport components of active zones and that they are tightly regulated.

  5. Deep recurrent conditional random field network for protein secondary prediction

    DEFF Research Database (Denmark)

    Johansen, Alexander Rosenberg; Sønderby, Søren Kaae; Sønderby, Casper Kaae

    2017-01-01

    Deep learning has become the state-of-the-art method for predicting protein secondary structure from only its amino acid residues and sequence profile. Building upon these results, we propose to combine a bi-directional recurrent neural network (biRNN) with a conditional random field (CRF), which...... of the labels for all time-steps. We condition the CRF on the output of biRNN, which learns a distributed representation based on the entire sequence. The biRNN-CRF is therefore close to ideally suited for the secondary structure task because a high degree of cross-talk between neighboring elements can...

  6. Protein Secondary Structure Prediction Using Deep Convolutional Neural Fields.

    Science.gov (United States)

    Wang, Sheng; Peng, Jian; Ma, Jianzhu; Xu, Jinbo

    2016-01-11

    Protein secondary structure (SS) prediction is important for studying protein structure and function. When only the sequence (profile) information is used as input feature, currently the best predictors can obtain ~80% Q3 accuracy, which has not been improved in the past decade. Here we present DeepCNF (Deep Convolutional Neural Fields) for protein SS prediction. DeepCNF is a Deep Learning extension of Conditional Neural Fields (CNF), which is an integration of Conditional Random Fields (CRF) and shallow neural networks. DeepCNF can model not only complex sequence-structure relationship by a deep hierarchical architecture, but also interdependency between adjacent SS labels, so it is much more powerful than CNF. Experimental results show that DeepCNF can obtain ~84% Q3 accuracy, ~85% SOV score, and ~72% Q8 accuracy, respectively, on the CASP and CAMEO test proteins, greatly outperforming currently popular predictors. As a general framework, DeepCNF can be used to predict other protein structure properties such as contact number, disorder regions, and solvent accessibility.

  7. Rab proteins: The key regulators of intracellular vesicle transport

    International Nuclear Information System (INIS)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-01-01

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

  8. Rab proteins: The key regulators of intracellular vesicle transport

    Energy Technology Data Exchange (ETDEWEB)

    Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  9. Identifying the molecular functions of electron transport proteins using radial basis function networks and biochemical properties.

    Science.gov (United States)

    Le, Nguyen-Quoc-Khanh; Nguyen, Trinh-Trung-Duong; Ou, Yu-Yen

    2017-05-01

    The electron transport proteins have an important role in storing and transferring electrons in cellular respiration, which is the most proficient process through which cells gather energy from consumed food. According to the molecular functions, the electron transport chain components could be formed with five complexes with several different electron carriers and functions. Therefore, identifying the molecular functions in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. This work includes two phases for discriminating electron transport proteins from transport proteins and classifying categories of five complexes in electron transport proteins. In the first phase, the performances from PSSM with AAIndex feature set were successful in identifying electron transport proteins in transport proteins with achieved sensitivity of 73.2%, specificity of 94.1%, and accuracy of 91.3%, with MCC of 0.64 for independent data set. With the second phase, our method can approach a precise model for identifying of five complexes with different molecular functions in electron transport proteins. The PSSM with AAIndex properties in five complexes achieved MCC of 0.51, 0.47, 0.42, 0.74, and 1.00 for independent data set, respectively. We suggest that our study could be a power model for determining new proteins that belongs into which molecular function of electron transport proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. VMD-SS: A graphical user interface plug-in to calculate the protein secondary structure in VMD program.

    Science.gov (United States)

    Yahyavi, Masoumeh; Falsafi-Zadeh, Sajad; Karimi, Zahra; Kalatarian, Giti; Galehdari, Hamid

    2014-01-01

    The investigation on the types of secondary structure (SS) of a protein is important. The evolution of secondary structures during molecular dynamics simulations is a useful parameter to analyze protein structures. Therefore, it is of interest to describe VMD-SS (a software program) for the identification of secondary structure elements and its trajectories during simulation for known structures available at the Protein Data Bank (PDB). The program helps to calculate (1) percentage SS, (2) SS occurrence in each residue, (3) percentage SS during simulation, and (4) percentage residues in all SS types during simulation. The VMD-SS plug-in was designed using TCL script and stride to calculate secondary structure features. The database is available for free at http://science.scu.ac.ir/HomePage.aspx?TabID=13755.

  11. Position-dependent Effects of Polylysine on Sec Protein Transport*

    Science.gov (United States)

    Liang, Fu-Cheng; Bageshwar, Umesh K.; Musser, Siegfried M.

    2012-01-01

    The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or “pause sites,” were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport. PMID:22367204

  12. Position-dependent effects of polylysine on Sec protein transport.

    Science.gov (United States)

    Liang, Fu-Cheng; Bageshwar, Umesh K; Musser, Siegfried M

    2012-04-13

    The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.

  13. Protein secondary structure prediction for a single-sequence using hidden semi-Markov models

    Directory of Open Access Journals (Sweden)

    Borodovsky Mark

    2006-03-01

    Full Text Available Abstract Background The accuracy of protein secondary structure prediction has been improving steadily towards the 88% estimated theoretical limit. There are two types of prediction algorithms: Single-sequence prediction algorithms imply that information about other (homologous proteins is not available, while algorithms of the second type imply that information about homologous proteins is available, and use it intensively. The single-sequence algorithms could make an important contribution to studies of proteins with no detected homologs, however the accuracy of protein secondary structure prediction from a single-sequence is not as high as when the additional evolutionary information is present. Results In this paper, we further refine and extend the hidden semi-Markov model (HSMM initially considered in the BSPSS algorithm. We introduce an improved residue dependency model by considering the patterns of statistically significant amino acid correlation at structural segment borders. We also derive models that specialize on different sections of the dependency structure and incorporate them into HSMM. In addition, we implement an iterative training method to refine estimates of HSMM parameters. The three-state-per-residue accuracy and other accuracy measures of the new method, IPSSP, are shown to be comparable or better than ones for BSPSS as well as for PSIPRED, tested under the single-sequence condition. Conclusions We have shown that new dependency models and training methods bring further improvements to single-sequence protein secondary structure prediction. The results are obtained under cross-validation conditions using a dataset with no pair of sequences having significant sequence similarity. As new sequences are added to the database it is possible to augment the dependency structure and obtain even higher accuracy. Current and future advances should contribute to the improvement of function prediction for orphan proteins inscrutable

  14. SAAS: Short Amino Acid Sequence - A Promising Protein Secondary Structure Prediction Method of Single Sequence

    Directory of Open Access Journals (Sweden)

    Zhou Yuan Wu

    2013-07-01

    Full Text Available In statistical methods of predicting protein secondary structure, many researchers focus on single amino acid frequencies in α-helices, β-sheets, and so on, or the impact near amino acids on an amino acid forming a secondary structure. But the paper considers a short sequence of amino acids (3, 4, 5 or 6 amino acids as integer, and statistics short sequence's probability forming secondary structure. Also, many researchers select low homologous sequences as statistical database. But this paper select whole PDB database. In this paper we propose a strategy to predict protein secondary structure using simple statistical method. Numerical computation shows that, short amino acids sequence as integer to statistics, which can easy see trend of short sequence forming secondary structure, and it will work well to select large statistical database (whole PDB database without considering homologous, and Q3 accuracy is ca. 74% using this paper proposed simple statistical method, but accuracy of others statistical methods is less than 70%.

  15. Combining sequence-based prediction methods and circular dichroism and infrared spectroscopic data to improve protein secondary structure determinations

    Directory of Open Access Journals (Sweden)

    Lees Jonathan G

    2008-01-01

    Full Text Available Abstract Background A number of sequence-based methods exist for protein secondary structure prediction. Protein secondary structures can also be determined experimentally from circular dichroism, and infrared spectroscopic data using empirical analysis methods. It has been proposed that comparable accuracy can be obtained from sequence-based predictions as from these biophysical measurements. Here we have examined the secondary structure determination accuracies of sequence prediction methods with the empirically determined values from the spectroscopic data on datasets of proteins for which both crystal structures and spectroscopic data are available. Results In this study we show that the sequence prediction methods have accuracies nearly comparable to those of spectroscopic methods. However, we also demonstrate that combining the spectroscopic and sequences techniques produces significant overall improvements in secondary structure determinations. In addition, combining the extra information content available from synchrotron radiation circular dichroism data with sequence methods also shows improvements. Conclusion Combining sequence prediction with experimentally determined spectroscopic methods for protein secondary structure content significantly enhances the accuracy of the overall results obtained.

  16. Multidrug and toxin extrusion proteins as transporters of antimicrobial drugs.

    Science.gov (United States)

    Nies, Anne T; Damme, Katja; Schaeffeler, Elke; Schwab, Matthias

    2012-12-01

    Antimicrobial drugs are essential in the treatment of infectious diseases. A better understanding of transport processes involved in drug disposition will improve the predictability of drug-drug interactions with consequences for drug response. Multidrug And Toxin Extrusion (MATE; SLC47A) proteins are efflux transporters mediating the excretion of several antimicrobial drugs as well as other organic compounds into bile and urine, thereby contributing to drug disposition. This review summarizes current knowledge of the structural and molecular features of human MATE transporters including their functional role in drug transport with a specific focus on antimicrobial drugs. The PubMed database was searched using the terms "MATE1," "MATE-2K," "MATE2," "SLC47A1," "SLC47A2," and "toxin extrusion protein" (up to June 2012). MATE proteins have been recognized as important transporters mediating the final excretion step of cationic drugs into bile and urine. These include the antiviral drugs acyclovir, amprenavir, and ganciclovir, the antibiotics cephalexin, cephradine and levofloxacin, as well as the antimalarial agents chloroquine and quinine. It is therefore important to enhance our understanding of the role of MATEs in drug extrusion with particular emphasis on the functional consequences of genetic variants on disposition of these antimicrobial drugs.

  17. Tungsten transport protein A (WtpA) in Pyrococcus furiosus: the first member of a new class of tungstate and molybdate transporters.

    Science.gov (United States)

    Bevers, Loes E; Hagedoorn, Peter-Leon; Krijger, Gerard C; Hagen, Wilfred R

    2006-09-01

    A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.

  18. Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements

    Energy Technology Data Exchange (ETDEWEB)

    Eghbalnia, Hamid R.; Wang Liya; Bahrami, Arash [National Magnetic Resonance Facility at Madison, Biochemistry Department (United States); Assadi, Amir [University of Wisconsin-Madison, Mathematics Department (United States); Markley, John L. [National Magnetic Resonance Facility at Madison, Biochemistry Department (United States)], E-mail: eghbalni@nmrfam.wisc.edu

    2005-05-15

    We present an energy model that combines information from the amino acid sequence of a protein and available NMR chemical shifts for the purposes of identifying low energy conformations and determining elements of secondary structure. The model ('PECAN', Protein Energetic Conformational Analysis from NMR chemical shifts) optimizes a combination of sequence information and residue-specific statistical energy function to yield energetic descriptions most favorable to predicting secondary structure. Compared to prior methods for secondary structure determination, PECAN provides increased accuracy and range, particularly in regions of extended structure. Moreover, PECAN uses the energetics to identify residues located at the boundaries between regions of predicted secondary structure that may not fit the stringent secondary structure class definitions. The energy model offers insights into the local energetic patterns that underlie conformational preferences. For example, it shows that the information content for defining secondary structure is localized about a residue and reaches a maximum when two residues on either side are considered. The current release of the PECAN software determines the well-defined regions of secondary structure in novel proteins with assigned chemical shifts with an overall accuracy of 90%, which is close to the practical limit of achievable accuracy in classifying the states.

  19. Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements

    International Nuclear Information System (INIS)

    Eghbalnia, Hamid R.; Wang Liya; Bahrami, Arash; Assadi, Amir; Markley, John L.

    2005-01-01

    We present an energy model that combines information from the amino acid sequence of a protein and available NMR chemical shifts for the purposes of identifying low energy conformations and determining elements of secondary structure. The model ('PECAN', Protein Energetic Conformational Analysis from NMR chemical shifts) optimizes a combination of sequence information and residue-specific statistical energy function to yield energetic descriptions most favorable to predicting secondary structure. Compared to prior methods for secondary structure determination, PECAN provides increased accuracy and range, particularly in regions of extended structure. Moreover, PECAN uses the energetics to identify residues located at the boundaries between regions of predicted secondary structure that may not fit the stringent secondary structure class definitions. The energy model offers insights into the local energetic patterns that underlie conformational preferences. For example, it shows that the information content for defining secondary structure is localized about a residue and reaches a maximum when two residues on either side are considered. The current release of the PECAN software determines the well-defined regions of secondary structure in novel proteins with assigned chemical shifts with an overall accuracy of 90%, which is close to the practical limit of achievable accuracy in classifying the states

  20. Loss of C. elegans BBS-7 and BBS-8 protein function results in cilia defects and compromised intraflagellar transport

    Science.gov (United States)

    Blacque, Oliver E.; Reardon, Michael J.; Li, Chunmei; McCarthy, Jonathan; Mahjoub, Moe R.; Ansley, Stephen J.; Badano, Jose L.; Mah, Allan K.; Beales, Philip L.; Davidson, William S.; Johnsen, Robert C.; Audeh, Mark; Plasterk, Ronald H.A.; Baillie, David L.; Katsanis, Nicholas; Quarmby, Lynne M.; Wicks, Stephen R.; Leroux, Michel R.

    2004-01-01

    Bardet-Biedl syndrome (BBS) is a genetically heterogeneous developmental disorder whose molecular basis is largely unknown. Here, we show that mutations in the Caenorhabditis elegans bbs-7 and bbs-8 genes cause structural and functional defects in cilia. C. elegans BBS proteins localize predominantly at the base of cilia, and like proteins involved in intraflagellar transport (IFT), a process necessary for cilia biogenesis and maintenance, move bidirectionally along the ciliary axoneme. Importantly, we demonstrate that BBS-7 and BBS-8 are required for the normal localization/motility of the IFT proteins OSM-5/Polaris and CHE-11, and to a notably lesser extent, CHE-2. We propose that BBS proteins play important, selective roles in the assembly and/or function of IFT particle components. Our findings also suggest that some of the cardinal and secondary symptoms of BBS, such as obesity, diabetes, cardiomyopathy, and learning defects may result from cilia dysfunction. PMID:15231740

  1. How cholesterol interacts with proteins and lipids during its intracellular transport

    DEFF Research Database (Denmark)

    Wüstner, Daniel; Solanko, Katarzyna

    2015-01-01

    as well as by non-vesicular sterol exchange between organelles. In this article, we will review recent progress in elucidating sterol-lipid and sterol-protein interactions contributing to proper sterol transport in living cells. We outline recent biophysical models of cholesterol distribution and dynamics...... for characterization of sterol-protein interactions and for monitoring intracellular sterol transport. Finally, we review recent work on the molecular mechanisms underlying lipoprotein-mediated cholesterol import into mammalian cells and describe the process of cellular cholesterol efflux. Overall, we emphasize how......Sterols, as cholesterol in mammalian cells and ergosterol in fungi, are indispensable molecules for proper functioning and nanoscale organization of the plasma membrane. Synthesis, uptake and efflux of cholesterol are regulated by a variety of protein-lipid and protein-protein interactions...

  2. Cysteine-rich intestinal protein binds zinc during transmucosal zinc transport

    International Nuclear Information System (INIS)

    Hempe, J.M.; Cousins, R.J.

    1991-01-01

    The mechanism of zinc absorption has not been delineated, but kinetic studies show that both passive and carrier-mediated processes are involved. The authors have identified a low molecular mass zinc-binding protein in the soluble fraction of rat intestinal mucosa that could function as an intracellular zinc carrier. The protein was not detected in liver or pancreas, suggesting a role specific to the intestine. The protein binds zinc during transmucosal zinc transport and shows signs of saturation at higher luminal zinc concentrations, characteristics consistent with a role in carrier-mediated zinc absorption. Microsequence analysis of the protein purified by gel-filtration HPCL and SDS/PAGE showed complete identity within the first 41 N-terminal amino acids with the deduced protein sequence of cysteine-rich intestinal protein. These investigators showed that the gene for this protein is developmentally regulated in neonates during the suckling period, conserved in many vertebrate species, and predominantly expressed in the small intestine. Cysteine-rich intestinal protein contains a recently identified conserved sequence of histidine and cysteine residues, the LIM motif, which our results suggest confers metal-binding properties that are important for zinc transport and/or functions of this micronutrient

  3. Improved protein structure reconstruction using secondary structures, contacts at higher distance thresholds, and non-contacts.

    Science.gov (United States)

    Adhikari, Badri; Cheng, Jianlin

    2017-08-29

    Residue-residue contacts are key features for accurate de novo protein structure prediction. For the optimal utilization of these predicted contacts in folding proteins accurately, it is important to study the challenges of reconstructing protein structures using true contacts. Because contact-guided protein modeling approach is valuable for predicting the folds of proteins that do not have structural templates, it is necessary for reconstruction studies to focus on hard-to-predict protein structures. Using a data set consisting of 496 structural domains released in recent CASP experiments and a dataset of 150 representative protein structures, in this work, we discuss three techniques to improve the reconstruction accuracy using true contacts - adding secondary structures, increasing contact distance thresholds, and adding non-contacts. We find that reconstruction using secondary structures and contacts can deliver accuracy higher than using full contact maps. Similarly, we demonstrate that non-contacts can improve reconstruction accuracy not only when the used non-contacts are true but also when they are predicted. On the dataset consisting of 150 proteins, we find that by simply using low ranked predicted contacts as non-contacts and adding them as additional restraints, can increase the reconstruction accuracy by 5% when the reconstructed models are evaluated using TM-score. Our findings suggest that secondary structures are invaluable companions of contacts for accurate reconstruction. Confirming some earlier findings, we also find that larger distance thresholds are useful for folding many protein structures which cannot be folded using the standard definition of contacts. Our findings also suggest that for more accurate reconstruction using predicted contacts it is useful to predict contacts at higher distance thresholds (beyond 8 Å) and predict non-contacts.

  4. Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

    NARCIS (Netherlands)

    Trip, Hein; Mulder, Niels L.; Lolkema, Juke S.

    Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells

  5. Structural basis of transport function in major facilitator superfamily protein from Trichoderma harzianum.

    Science.gov (United States)

    Chaudhary, Nitika; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2017-02-01

    Trichothecenes are the sesquiterpenes secreted by Trichoderma spp. residing in the rhizosphere. These compounds have been reported to act as plant growth promoters and bio-control agents. The structural knowledge for the transporter proteins of their efflux remained limited. In this study, three-dimensional structure of Thmfs1 protein, a trichothecene transporter from Trichoderma harzianum, was homology modelled and further Molecular Dynamics (MD) simulations were used to decipher its mechanism. Fourteen transmembrane helices of Thmfs1 protein are observed contributing to an inward-open conformation. The transport channel and ligand binding sites in Thmfs1 are identified based on heuristic, iterative algorithm and structural alignment with homologous proteins. MD simulations were performed to reveal the differential structural behaviour occurring in the ligand free and ligand bound forms. We found that two discrete trichothecene binding sites are located on either side of the central transport tunnel running from the cytoplasmic side to the extracellular side across the Thmfs1 protein. Detailed analysis of the MD trajectories showed an alternative access mechanism between N and C-terminal domains contributing to its function. These results also demonstrate that the transport of trichodermin occurs via hopping mechanism in which the substrate molecule jumps from one binding site to another lining the transport tunnel. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. The secondary structure and the thermal unfolding parameters of the S-layer protein from Lactobacillus salivarius.

    Science.gov (United States)

    Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian

    2016-09-01

    Surface layer (S-layer) proteins have been identified in the cell envelope of many organisms, such as bacteria and archaea. They self-assemble, forming monomolecular crystalline arrays. Isolated S-layer proteins are able to recrystallize into regular lattices, which proved useful in biotechnology. Here we investigate the structure and thermal unfolding of the S-layer protein isolated from Lactobacillus salivarius 16 strain of human origin. Using circular dichroism (CD) spectroscopy, and the software CDSSTR from DICHROWEB, CONTINLL from CDPro, as well as CDNN, we assess the fractions of the protein's secondary structural elements at temperatures ranging between 10 and 90 °C, and predict the tertiary class of the protein. To study the thermal unfolding of the protein, we analyze the temperature dependence of the CD signal in the far- and near-UV domains. Fitting the experimental data by two- and three-state models of thermal unfolding, we infer the midpoint temperatures, the temperature dependence of the changes in Gibbs free energy, enthalpy, and entropy of the unfolding transitions in standard conditions, and the temperature dependence of the equilibrium constant. We also estimate the changes in heat capacity at constant pressure in standard conditions. The results indicate that the thermal unfolding of the S-layer protein from L. salivarius is highly cooperative, since changes in the secondary and tertiary structures occur simultaneously. The thermodynamic analysis predicts a "cold" transition, at about -3 °C, of both the secondary and tertiary structures. Our findings may be important for the use of S-layer proteins in biotechnology and in biomedical applications.

  7. Tubule urate and PAH transport: sensitivity and specificity of serum protein inhibition

    International Nuclear Information System (INIS)

    Grantham, J.J.; Kennedy, J.; Cowley, B.

    1987-01-01

    Macromolecules in rabbit serum inhibit the cellular uptake and transepithelial secretion of [ 14 C]urate and p-[ 3 H]aminohippurate ([ 3 H]PAH) in rabbit S 2 proximal tubule segments. To understand better the potential role these inhibitors may have in the regulation of renal organic anion excretion, the authors examined the specificity and relative inhibitory effects on tubule urate and PAH transport of albumin and γ-globulin, the major inhibitory proteins in rabbit serum. Native rabbit serum markedly inhibited the cellular accumulation or urate and PAH by isolated nonperfused segments. Urate and PAH transport was also inhibited by bovine serum, human serum, Cohn-fractionated rabbit albumin, and rabbit γ-globulin, but not by Cohn-fractionated bovine serum albumin. α-Lactalbumin and β-lactoglobulin, derived from milk, also inhibited urate and PAH transport, but to a lesser extent than albumin and γ-globulin. The transport inhibitory effects of proteins were independent of their binding to urate and PAH. Unidirectional influx and the steady-state intracellular accumulation of urate and PAH in suspensions of proximal tubules were decreased by rabbit serum proteins, suggesting that these inhibitors act on the external face of the cells to diminish the uptake of the organic anions. These studies indicate that the principal plasma proteins (albumin and γ-globulin) significantly inhibit urate and PAH transporters in the basolateral membranes of S 2 proximal tubules. They suggest that circulating plasma proteins that can penetrate the basement membrane of proximal tubules may directly modulate the renal excretion of urate and PAH

  8. Evaluation of all-electric secondary power for transport aircraft

    Science.gov (United States)

    Murray, W. E.; Feiner, L. J.; Flores, R. R.

    1992-01-01

    This report covers a study by Douglas Aircraft Company (DAC) of electrical power systems for advanced transport aircraft based upon an all-electric design concept. The concept would eliminate distributed hydraulic and pneumatic secondary power systems, and feature an expanded secondary electrical power system redesigned to supply power to the loads customarily supplied by hydraulic or pneumatic power. The initial study was based on an advanced 20-kHz electrical power transmission and distribution system, using a system architecture supplied by NASA-Lewis Research Center for twin-engine aircraft with many advanced power conversion concepts. NASA-LeRC later requested DAC to refocus the study on 400-Hz secondary power distribution. Subsequent work was based on a three-engine MD-11 aircraft, selected by DAC as a baseline system design that would provide data for the comparative cost/benefit analysis. The study concluded that the 20-kHz concept produced many expected benefits, and that the all-electric trijet weight savings on hardware redesign would be 2,304 pounds plus a 2.1-percent fuel reduction and resized for a total weight reduction of 11,000 pounds. Cost reductions for a fleet of 800 aircraft in a 15-year production program were estimated at $76.71 million for RDT&E; $2.74 million per aircrat for production; $9.84 million for nonrecurring expenses; $120,000 per aircraft for product support; and $300,000 per aircraft per year for operating and maintenance costs, giving a present value of $1.914 billion saved or a future value of $10.496 billion saved.

  9. CNNH_PSS: protein 8-class secondary structure prediction by convolutional neural network with highway.

    Science.gov (United States)

    Zhou, Jiyun; Wang, Hongpeng; Zhao, Zhishan; Xu, Ruifeng; Lu, Qin

    2018-05-08

    Protein secondary structure is the three dimensional form of local segments of proteins and its prediction is an important problem in protein tertiary structure prediction. Developing computational approaches for protein secondary structure prediction is becoming increasingly urgent. We present a novel deep learning based model, referred to as CNNH_PSS, by using multi-scale CNN with highway. In CNNH_PSS, any two neighbor convolutional layers have a highway to deliver information from current layer to the output of the next one to keep local contexts. As lower layers extract local context while higher layers extract long-range interdependencies, the highways between neighbor layers allow CNNH_PSS to have ability to extract both local contexts and long-range interdependencies. We evaluate CNNH_PSS on two commonly used datasets: CB6133 and CB513. CNNH_PSS outperforms the multi-scale CNN without highway by at least 0.010 Q8 accuracy and also performs better than CNF, DeepCNF and SSpro8, which cannot extract long-range interdependencies, by at least 0.020 Q8 accuracy, demonstrating that both local contexts and long-range interdependencies are indeed useful for prediction. Furthermore, CNNH_PSS also performs better than GSM and DCRNN which need extra complex model to extract long-range interdependencies. It demonstrates that CNNH_PSS not only cost less computer resource, but also achieves better predicting performance. CNNH_PSS have ability to extracts both local contexts and long-range interdependencies by combing multi-scale CNN and highway network. The evaluations on common datasets and comparisons with state-of-the-art methods indicate that CNNH_PSS is an useful and efficient tool for protein secondary structure prediction.

  10. RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Dabney-Smith, Carole [Miami Univ., Oxford, OH (United States)

    2016-07-18

    Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembled proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cp

  11. Influence of secondary structure on in-source decay of protein in matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Takayama, Mitsuo; Osaka, Issey; Sakakura, Motoshi

    2012-01-01

    The susceptibility of the N-Cα bond of the peptide backbone to specific cleavage by in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was studied from the standpoint of the secondary structure of three proteins. A naphthalene derivative, 5-amino-1-naphtol (5,1-ANL), was used as the matrix. The resulting c'-ions, which originate from the cleavage at N-Cα bonds in flexible secondary structures such as turn and bend, and are free from intra-molecular hydrogen-bonded α-helix structure, gave relatively intense peaks. Furthermore, ISD spectra of the proteins showed that the N-Cα bonds of specific amino acid residues, namely Gly-Xxx, Xxx-Asp, and Xxx-Asn, were more susceptible to MALDI-ISD than other amino acid residues. This is in agreement with the observation that Gly, Asp and Asn residues usually located in turns, rather than α-helix. The results obtained indicate that protein molecules embedded into the matrix crystal in the MALDI experiments maintain their secondary structures as determined by X-ray crystallography, and that MALDI-ISD has the capability for providing information concerning the secondary structure of protein.

  12. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    Science.gov (United States)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  13. Secondary electron emission and self-consistent charge transport in semi-insulating samples

    Energy Technology Data Exchange (ETDEWEB)

    Fitting, H.-J. [Institute of Physics, University of Rostock, Universitaetsplatz 3, D-18051 Rostock (Germany); Touzin, M. [Unite Materiaux et Transformations, UMR CNRS 8207, Universite de Lille 1, F-59655 Villeneuve d' Ascq (France)

    2011-08-15

    Electron beam induced self-consistent charge transport and secondary electron emission (SEE) in insulators are described by means of an electron-hole flight-drift model (FDM) now extended by a certain intrinsic conductivity (c) and are implemented by an iterative computer simulation. Ballistic secondary electrons (SE) and holes, their attenuation to drifting charge carriers, and their recombination, trapping, and field- and temperature-dependent detrapping are included. As a main result the time dependent ''true'' secondary electron emission rate {delta}(t) released from the target material and based on ballistic electrons and the spatial distributions of currents j(x,t), charges {rho}(x,t), field F(x,t), and potential V(x,t) are obtained where V{sub 0} = V(0,t) presents the surface potential. The intrinsic electronic conductivity limits the charging process and leads to a conduction sample current to the support. In that case the steady-state total SE yield will be fixed below the unit: i.e., {sigma} {eta} + {delta} < 1.

  14. Parameterized Flight Mission for Secondary Power Requirement Estimations of Commercial Transport Aircraft

    OpenAIRE

    Lampl, Thomas; Muschkorgel, Sandra; Hornung, Mirko;

    2018-01-01

    The trend towards More-Electric Aircraft (MEA) and the introduction of new system technologies lead to considerable changes at the system level of commercial transport aircraft. Because the number of systems and power requirements are increasing, the consideration and integration of aircraft systems in early aircraft design phases is important. The objective of this contribution is to develop a characteristic flight mission with modelled aircraft systems to estimate the secondary power requir...

  15. CSSI-PRO: a method for secondary structure type editing, assignment and estimation in proteins using linear combination of backbone chemical shifts

    International Nuclear Information System (INIS)

    Swain, Monalisa; Atreya, Hanudatta S.

    2009-01-01

    Estimation of secondary structure in polypeptides is important for studying their structure, folding and dynamics. In NMR spectroscopy, such information is generally obtained after sequence specific resonance assignments are completed. We present here a new methodology for assignment of secondary structure type to spin systems in proteins directly from NMR spectra, without prior knowledge of resonance assignments. The methodology, named Combination of Shifts for Secondary Structure Identification in Proteins (CSSI-PRO), involves detection of specific linear combination of backbone 1 H α and 13 C' chemical shifts in a two-dimensional (2D) NMR experiment based on G-matrix Fourier transform (GFT) NMR spectroscopy. Such linear combinations of shifts facilitate editing of residues belonging to α-helical/β-strand regions into distinct spectral regions nearly independent of the amino acid type, thereby allowing the estimation of overall secondary structure content of the protein. Comparison of the predicted secondary structure content with those estimated based on their respective 3D structures and/or the method of Chemical Shift Index for 237 proteins gives a correlation of more than 90% and an overall rmsd of 7.0%, which is comparable to other biophysical techniques used for structural characterization of proteins. Taken together, this methodology has a wide range of applications in NMR spectroscopy such as rapid protein structure determination, monitoring conformational changes in protein-folding/ligand-binding studies and automated resonance assignment

  16. Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

    Science.gov (United States)

    Beznoussenko, Galina V; Parashuraman, Seetharaman; Rizzo, Riccardo; Polishchuk, Roman; Martella, Oliviano; Di Giandomenico, Daniele; Fusella, Aurora; Spaar, Alexander; Sallese, Michele; Capestrano, Maria Grazia; Pavelka, Margit; Vos, Matthijn R; Rikers, Yuri GM; Helms, Volkhard; Mironov, Alexandre A; Luini, Alberto

    2014-01-01

    The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression–maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes. DOI: http://dx.doi.org/10.7554/eLife.02009.001 PMID:24867214

  17. Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

    Science.gov (United States)

    Finkenwirth, Friedrich; Kirsch, Franziska

    2013-01-01

    Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. PMID:23836870

  18. Water-transporting proteins

    DEFF Research Database (Denmark)

    Zeuthen, Thomas

    2010-01-01

    . In the K(+)/Cl(-) and the Na(+)/K(+)/2Cl(-) cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na(+)-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water...... transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support...... to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity...

  19. The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.

    Science.gov (United States)

    Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

    2015-01-09

    Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. The Putative Cellodextrin Transporter-like Protein CLP1 Is Involved in Cellulase Induction in Neurospora crassa*

    Science.gov (United States)

    Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

    2015-01-01

    Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. PMID:25398875

  1. Modelling Transcapillary Transport of Fluid and Proteins in Hemodialysis Patients.

    Directory of Open Access Journals (Sweden)

    Mauro Pietribiasi

    Full Text Available The kinetics of protein transport to and from the vascular compartment play a major role in the determination of fluid balance and plasma refilling during hemodialysis (HD sessions. In this study we propose a whole-body mathematical model describing water and protein shifts across the capillary membrane during HD and compare its output to clinical data while evaluating the impact of choosing specific values for selected parameters.The model follows a two-compartment structure (vascular and interstitial space and is based on balance equations of protein mass and water volume in each compartment. The capillary membrane was described according to the three-pore theory. Two transport parameters, the fractional contribution of large pores (αLP and the total hydraulic conductivity (LpS of the capillary membrane, were estimated from patient data. Changes in the intensity and direction of individual fluid and solute flows through each part of the transport system were analyzed in relation to the choice of different values of small pores radius and fractional conductivity, lymphatic sensitivity to hydraulic pressure, and steady-state interstitial-to-plasma protein concentration ratio.The estimated values of LpS and αLP were respectively 10.0 ± 8.4 mL/min/mmHg (mean ± standard deviation and 0.062 ± 0.041. The model was able to predict with good accuracy the profiles of plasma volume and serum total protein concentration in most of the patients (average root-mean-square deviation < 2% of the measured value.The applied model provides a mechanistic interpretation of fluid transport processes induced by ultrafiltration during HD, using a minimum of tuned parameters and assumptions. The simulated values of individual flows through each kind of pore and lymphatic absorption rate yielded by the model may suggest answers to unsolved questions on the relative impact of these not-measurable quantities on total vascular refilling and fluid balance.

  2. Evaluation of secondary exposure doses to transportation and medical personnel in the radiation emergency medicine

    International Nuclear Information System (INIS)

    Kato, Hideki; Suzuki, Shoichi; Koga, Sukehiko; Mukoyama, Takashi; Tomatsu, Hirotaka; Suzuki, Yusuke

    2009-01-01

    Radionuclide contamination is one of the accidents that might occur while carrying out a periodical inspection of nuclear power stations during normal reactor operation. When such an accident occurs, rescue and medical personnel, involved in transporting and treating affected workers run the risk of exposure to secondary radiation. In this study, the ambient dose equivalent rate at a certain distance from the surface of the human body contaminated with typical radioactive corrosion products was calculated. Further, the relationships among the adhesion area, contamination density, and secondary exposure dose were clarified. The secondary exposure dose and permissible working hours in a radiation emergency medicine were estimated by presenting these relationships in the form of a chart and by specifying the contamination levels. (author)

  3. Intracellular Transport and Kinesin Superfamily Proteins: Structure, Function and Dynamics

    Science.gov (United States)

    Hirokawa, N.; Takemura, R.

    Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

  4. Electronic transport on the spatial structure of the protein: Three-dimensional lattice model

    International Nuclear Information System (INIS)

    Sarmento, R.G.; Frazão, N.F.; Macedo-Filho, A.

    2017-01-01

    Highlights: • The electronic transport on the structure of the three-dimensional lattice model of the protein is studied. • The signing of the current–voltage is directly affected by permutations of the weak bonds in the structure. • Semiconductor behave of the proteins suggest a potential application in the development of novel biosensors. - Abstract: We report a numerical analysis of the electronic transport in protein chain consisting of thirty-six standard amino acids. The protein chains studied have three-dimensional structure, which can present itself in three distinct conformations and the difference consist in the presence or absence of thirteen hydrogen-bondings. Our theoretical method uses an electronic tight-binding Hamiltonian model, appropriate to describe the protein segments modeled by the amino acid chain. We note that the presence and the permutations between weak bonds in the structure of proteins are directly related to the signing of the current–voltage. Furthermore, the electronic transport depends on the effect of temperature. In addition, we have found a semiconductor behave in the models investigated and it suggest a potential application in the development of novel biosensors for molecular diagnostics.

  5. Electronic transport on the spatial structure of the protein: Three-dimensional lattice model

    Energy Technology Data Exchange (ETDEWEB)

    Sarmento, R.G. [Departamento de Ciências Biológicas, Universidade Federal do Piauí, 64800-000 Floriano, PI (Brazil); Frazão, N.F. [Centro de Educação e Saúde, Universidade Federal de Campina Grande, 581750-000 Cuité, PB (Brazil); Macedo-Filho, A., E-mail: amfilho@gmail.com [Campus Prof. Antonio Geovanne Alves de Sousa, Universidade Estadual do Piauí, 64260-000 Piripiri, PI (Brazil)

    2017-01-30

    Highlights: • The electronic transport on the structure of the three-dimensional lattice model of the protein is studied. • The signing of the current–voltage is directly affected by permutations of the weak bonds in the structure. • Semiconductor behave of the proteins suggest a potential application in the development of novel biosensors. - Abstract: We report a numerical analysis of the electronic transport in protein chain consisting of thirty-six standard amino acids. The protein chains studied have three-dimensional structure, which can present itself in three distinct conformations and the difference consist in the presence or absence of thirteen hydrogen-bondings. Our theoretical method uses an electronic tight-binding Hamiltonian model, appropriate to describe the protein segments modeled by the amino acid chain. We note that the presence and the permutations between weak bonds in the structure of proteins are directly related to the signing of the current–voltage. Furthermore, the electronic transport depends on the effect of temperature. In addition, we have found a semiconductor behave in the models investigated and it suggest a potential application in the development of novel biosensors for molecular diagnostics.

  6. Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2009-06-01

    Full Text Available Abstract Background Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae. Results To investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H+/glycerol transporters. Conclusion This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org.

  7. Proteins Related to the Type I Secretion System Are Associated with Secondary SecA_DEAD Domain Proteins in Some Species of Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi.

    Directory of Open Access Journals (Sweden)

    Olga K Kamneva

    Full Text Available A number of bacteria belonging to the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae super-phylum contain unusual ribosome-bearing intracellular membranes. The evolutionary origins and functions of these membranes are unknown. Some proteins putatively associated with the presence of intracellular membranes in PVC bacteria contain signal peptides. Signal peptides mark proteins for translocation across the cytoplasmic membrane in prokaryotes, and the membrane of the endoplasmic reticulum in eukaryotes, by highly conserved Sec machinery. This suggests that proteins might be targeted to intracellular membranes in PVC bacteria via the Sec pathway. Here, we show that canonical signal peptides are significantly over-represented in proteins preferentially present in PVC bacteria possessing intracellular membranes, indicating involvement of Sec translocase in their cellular targeting. We also characterized Sec proteins using comparative genomics approaches, focusing on the PVC super-phylum. While we were unable to detect unique changes in Sec proteins conserved among membrane-bearing PVC species, we identified (1 SecA ATPase domain re-arrangements in some Planctomycetes, and (2 secondary SecA_DEAD domain proteins in the genomes of some Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi. This is the first report of potentially duplicated SecA in Gram-negative bacteria. The phylogenetic distribution of secondary SecA_DEAD domain proteins suggests that the presence of these proteins is not related to the occurrence of PVC endomembranes. Further genomic analysis showed that secondary SecA_DEAD domain proteins are located within genomic neighborhoods that also encode three proteins possessing domains specific for the Type I secretion system.

  8. The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

    Energy Technology Data Exchange (ETDEWEB)

    Koropatkin, Nicole M.; Koppenaal, David W.; Pakrasi, Himadri B.; Smith, Thomas J.

    2007-01-26

    Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO₂ over O₂, Cyanobacteria have developed highly efficient CO₂concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here we describe the structure of the bicarbonate binding protein, CmpA, in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein, NrtA. CmpA binds carbonic acid at the entrance to the ligand-binding pocket whereas bicarbonate binds in nearly an identical location compared to nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca²⁺ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal is highly cooperative and suggests that CmpA co-transports bicarbonate and calcium.

  9. The alternating access mechanism of transport as observed in the sodium-hydantoin transporter Mhp1

    International Nuclear Information System (INIS)

    Weyand, Simone; Shimamura, Tatsuro; Beckstein, Oliver; Sansom, Mark S. P.; Iwata, So; Henderson, Peter J. F.; Cameron, Alexander D.

    2011-01-01

    Crystal structures of a membrane protein transporter in three different conformational states provide insights into the transport mechanism. Secondary active transporters move molecules across cell membranes by coupling this process to the energetically favourable downhill movement of ions or protons along an electrochemical gradient. They function by the alternating access model of transport in which, through conformational changes, the substrate binding site alternately faces either side of the membrane. Owing to the difficulties in obtaining the crystal structure of a single transporter in different conformational states, relatively little structural information is known to explain how this process occurs. Here, the structure of the sodium-benzylhydantoin transporter, Mhp1, from Microbacterium liquefaciens, has been determined in three conformational states; from this a mechanism is proposed for switching from the outward-facing open conformation through an occluded structure to the inward-facing open state

  10. Ion Binding Energies Determining Functional Transport of ClC Proteins

    Science.gov (United States)

    Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

    2014-06-01

    The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

  11. A solute-binding protein for iron transport in Streptococcus iniae

    Directory of Open Access Journals (Sweden)

    Li Anxing

    2010-12-01

    Full Text Available Abstract Background Streptococcus iniae (S. iniae is a major pathogen that causes considerable morbidity and mortality in cultured fish worldwide. The pathogen's ability to adapt to the host affects the extent of infection, hence understanding the mechanisms by which S. iniae overcomes physiological stresses during infection will help to identify potential virulence determinants of streptococcal infection. Grow S. iniae under iron-restricted conditions is one approach for identifying host-specific protein expression. Iron plays an important role in many biological processes but it has low solubility under physiological condition. Many microorganisms have been shown to be able to circumvent this nutritional limitation by forming direct contacts with iron-containing proteins through ATP-binding cassette (ABC transporters. The ABC transporter superfamilies constitute many different systems that are widespread among living organisms with different functions, such as ligands translocation, mRNA translation, and DNA repair. Results An ABC transporter system, named as mtsABC (metal transport system was cloned from S. iniae HD-1, and was found to be involved in heme utilization. mtsABC is cotranscribed by three downstream genes, i.e., mtsA, mtsB, and mtsC. In this study, we cloned the first gene of the mtsABC transporter system (mtsA, and purified the corresponding recombinant protein MtsA. The analysis indicated that MtsA is a putative lipoprotein which binds to heme that can serve as an iron source for the microorganism, and is expressed in vivo during Kunming mice infection by S. iniae HD-1. Conclusions This is believed to be the first report on the cloning the ABC transporter lipoprotein from S. iniae genomic DNA. Together, our data suggested that MtsA is associated with heme, and is expressed in vivo during Kunming mice infection by S. iniae HD-1 which indicated that it can be a potential candidate for S. iniae subunit vaccine.

  12. Improved age-diffusion model for low-energy electron transport in solids. II. Application to secondary emission from aluminum

    International Nuclear Information System (INIS)

    Dubus, A.; Devooght, J.; Dehaes, J.C.

    1987-01-01

    The ''improved age-diffusion'' model for secondary-electron transport is applied to aluminum. Electron cross sections for inelastic collisions with the free-electron gas using the Lindhard dielectric function and for elastic collisions with the randomly distributed ionic cores are used in the calculations. The most important characteristics of backward secondary-electron emission induced by low-energy electrons on polycrystalline Al targets are calculated and compared to experimental results and to Monte Carlo calculations. The model appears to predict the electronic yield, the energy spectra, and the spatial dependence of secondary emission with reasonable accuracy

  13. Uptake and transport of manganese in primary and secondary olfactory neurones in pike.

    Science.gov (United States)

    Tjälve, H; Mejàre, C; Borg-Neczak, K

    1995-07-01

    gamma-spectrometry and autoradiography were used to examine the axoplasmic flow of manganese in the olfactory nerves and to study the uptake of the metal in the brain after application of 54Mn2+ in the olfactory chambers of pikes. The results show that the 54Mn2+ is taken up in the olfactory receptor cells and is transported at a constant rate along the primary olfactory neurones into the brain. The maximal velocity for the transported 54Mn2+ was 2.90 +/- 0.21 mm/hr (mean +/- S.E.) at 10 degrees, which was the temperature used in the experiments. The 54Mn2+ accumulated in the entire olfactory bulbs, although most marked in central and caudal parts. The metal was also seen to migrate into large areas of the telencephalon, apparently mainly via the secondary olfactory axons present in the medial olfactory tract. A transfer along fibres of the medial olfactory tract probably also explains the labelling which was seen in the diencephalon down to the hypothalamus. The results also showed that there is a pathway connecting the two olfactory bulbs of the pike and that this can carry the metal. Our data further showed a marked accumulation of 54Mn2+ in the meningeal epithelium and in the contents of the meningeal sacs surrounding the olfactory bulbs. It appears from our study that manganese has the ability to pass the synaptic junctions between the primary and the secondary olfactory neurones in the olfactory bulbs and to migrate along secondary olfactory pathways into the telencephalon and the diencephalon.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Protein 8-class secondary structure prediction using conditional neural fields.

    Science.gov (United States)

    Wang, Zhiyong; Zhao, Feng; Peng, Jian; Xu, Jinbo

    2011-10-01

    Compared with the protein 3-class secondary structure (SS) prediction, the 8-class prediction gains less attention and is also much more challenging, especially for proteins with few sequence homologs. This paper presents a new probabilistic method for 8-class SS prediction using conditional neural fields (CNFs), a recently invented probabilistic graphical model. This CNF method not only models the complex relationship between sequence features and SS, but also exploits the interdependency among SS types of adjacent residues. In addition to sequence profiles, our method also makes use of non-evolutionary information for SS prediction. Tested on the CB513 and RS126 data sets, our method achieves Q8 accuracy of 64.9 and 64.7%, respectively, which are much better than the SSpro8 web server (51.0 and 48.0%, respectively). Our method can also be used to predict other structure properties (e.g. solvent accessibility) of a protein or the SS of RNA. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Study of the transport of mercurial compounds by seric proteins

    International Nuclear Information System (INIS)

    Jullien-Saint Guily, Nicole

    1970-01-01

    A bond between the seric proteins and various mercurial compounds labeled with the radioisotopes 203 Hg and 197 Hg was demonstrated by means of research methods specific to radioactivity combined with protein separation techniques. In the course of this study it was shown how strongly the composition of the buffer during electrophoretic migration influences the transport of certain organo-mercurial compounds by the seric proteins. By means of a thioloprive: N - ethyl - maleimide, labeled with 14 C, it was proved that the bonding sites between the proteins and the mercurial compounds were the thiol groups of the proteins but that other bonding sites, in particular the amino groups, could also be involved. (author) [fr

  16. Secondary Structure Preferences of Mn2+ Binding Sites in Bacterial Proteins

    Directory of Open Access Journals (Sweden)

    Tatyana Aleksandrovna Khrustaleva

    2014-01-01

    Full Text Available 3D structures of proteins with coordinated Mn2+ ions from bacteria with low, average, and high genomic GC-content have been analyzed (149 PDB files were used. Major Mn2+ binders are aspartic acid (6.82% of Asp residues, histidine (14.76% of His residues, and glutamic acid (3.51% of Glu residues. We found out that the motif of secondary structure “beta strand-major binder-random coil” is overrepresented around all the three major Mn2+ binders. That motif may be followed by either alpha helix or beta strand. Beta strands near Mn2+ binding residues should be stable because they are enriched by such beta formers as valine and isoleucine, as well as by specific combinations of hydrophobic and hydrophilic amino acid residues characteristic to beta sheet. In the group of proteins from GC-rich bacteria glutamic acid residues situated in alpha helices frequently coordinate Mn2+ ions, probably, because of the decrease of Lys usage under the influence of mutational GC-pressure. On the other hand, the percentage of Mn2+ sites with at least one amino acid in the “beta strand-major binder-random coil” motif of secondary structure (77.88% does not depend on genomic GC-content.

  17. Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Jørgensen, Trine Nygaard; Gether, Ulrik

    2010-01-01

    -synaptic neurons. This has led to the identification of a plethora of different kinases, receptors and scaffolding proteins that interact with DAT and hereby either modulate the catalytic activity of the transporter or regulate its trafficking and degradation. Several new tools for studying DAT regulation in live...

  18. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  19. Yarrowia lipolytica vesicle-mediated protein transport pathways

    Directory of Open Access Journals (Sweden)

    Beckerich Jean-Marie

    2007-11-01

    transport shows that 40% of Y. lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S. cerevisiae. Conclusion These results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y. lipolytica is more representative of vesicular secretion of animals and other fungi than is S. cerevisiae.

  20. Protein secondary structure assignment revisited: a detailed analysis of different assignment methods

    Directory of Open Access Journals (Sweden)

    de Brevern Alexandre G

    2005-09-01

    Full Text Available Abstract Background A number of methods are now available to perform automatic assignment of periodic secondary structures from atomic coordinates, based on different characteristics of the secondary structures. In general these methods exhibit a broad consensus as to the location of most helix and strand core segments in protein structures. However the termini of the segments are often ill-defined and it is difficult to decide unambiguously which residues at the edge of the segments have to be included. In addition, there is a "twilight zone" where secondary structure segments depart significantly from the idealized models of Pauling and Corey. For these segments, one has to decide whether the observed structural variations are merely distorsions or whether they constitute a break in the secondary structure. Methods To address these problems, we have developed a method for secondary structure assignment, called KAKSI. Assignments made by KAKSI are compared with assignments given by DSSP, STRIDE, XTLSSTR, PSEA and SECSTR, as well as secondary structures found in PDB files, on 4 datasets (X-ray structures with different resolution range, NMR structures. Results A detailed comparison of KAKSI assignments with those of STRIDE and PSEA reveals that KAKSI assigns slightly longer helices and strands than STRIDE in case of one-to-one correspondence between the segments. However, KAKSI tends also to favor the assignment of several short helices when STRIDE and PSEA assign longer, kinked, helices. Helices assigned by KAKSI have geometrical characteristics close to those described in the PDB. They are more linear than helices assigned by other methods. The same tendency to split long segments is observed for strands, although less systematically. We present a number of cases of secondary structure assignments that illustrate this behavior. Conclusion Our method provides valuable assignments which favor the regularity of secondary structure segments.

  1. Complement activation by ceramide transporter proteins.

    Science.gov (United States)

    Bode, Gerard H; Losen, Mario; Buurman, Wim A; Veerhuis, Robert; Molenaar, Peter C; Steinbusch, Harry W M; De Baets, Marc H; Daha, Mohamed R; Martinez-Martinez, Pilar

    2014-02-01

    C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with extracellular matrix components, such as type IV collagen, and with the innate immune protein serum amyloid P. In this article, we report a novel function of CERT in the innate immune response. Both CERT isoforms, when immobilized, were found to bind the globular head region of C1q and to initiate the classical complement pathway, leading to activation of C4 and C3, as well as generation of the membrane attack complex C5b-9. In addition, C1q was shown to bind to endogenous CERTL on the surface of apoptotic cells. These results demonstrate the role of CERTs in innate immunity, especially in the clearance of apoptotic cells.

  2. Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter.

    OpenAIRE

    Neyfakh, A A; Borsch, C M; Kaatz, G W

    1993-01-01

    The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

  3. Aquaporin-11: A channel protein lacking apparent transport function expressed in brain

    Directory of Open Access Journals (Sweden)

    Tsunenari Takashi

    2006-05-01

    Full Text Available Abstract Background The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. Results We report on the characterization of Aquaporin-11 (AQP11. AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. Conclusion AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain.

  4. A novel Multi-Agent Ada-Boost algorithm for predicting protein structural class with the information of protein secondary structure.

    Science.gov (United States)

    Fan, Ming; Zheng, Bin; Li, Lihua

    2015-10-01

    Knowledge of the structural class of a given protein is important for understanding its folding patterns. Although a lot of efforts have been made, it still remains a challenging problem for prediction of protein structural class solely from protein sequences. The feature extraction and classification of proteins are the main problems in prediction. In this research, we extended our earlier work regarding these two aspects. In protein feature extraction, we proposed a scheme by calculating the word frequency and word position from sequences of amino acid, reduced amino acid, and secondary structure. For an accurate classification of the structural class of protein, we developed a novel Multi-Agent Ada-Boost (MA-Ada) method by integrating the features of Multi-Agent system into Ada-Boost algorithm. Extensive experiments were taken to test and compare the proposed method using four benchmark datasets in low homology. The results showed classification accuracies of 88.5%, 96.0%, 88.4%, and 85.5%, respectively, which are much better compared with the existing methods. The source code and dataset are available on request.

  5. Which Socio-Ecological Factors Associate with a Switch to or Maintenance of Active and Passive Transport during the Transition from Primary to Secondary School?

    Science.gov (United States)

    Vanwolleghem, Griet; Van Dyck, Delfien; De Meester, Femke; De Bourdeaudhuij, Ilse; Cardon, Greet; Gheysen, Freja

    2016-01-01

    The aim was to investigate which individual, psychosocial and physical neighborhood environmental factors associate with children's switch to or maintenance of active/passive transport to school and to leisure time destinations during the transition from primary to secondary school. Children (n = 313) filled out a questionnaire in the last year of primary school and 2 years later to assess socio-demographic characteristics and self-reported transport. One of their parents completed a questionnaire to assess parental perceptions of psychosocial and physical neighborhood environmental factors. The increase of the home-school distance was significantly associated with children's switch to or maintenance of passive transport to school compared to a switch to (OR = 0.81; p = 0.03) and maintenance (OR = 0.87; p = 0.03) of active transport to school. Low SES was associated with children's switch to active transport to school compared to maintenance of active transport (OR = 3.67; p = 0.07). For transport to leisure time destinations, other factors such as parental perceived neighborhood safety from traffic and crime (OR = 2.78; p = 0.004), a positive social norm (OR = 1.49; p = 0.08), positive attitudes (OR = 1.39; p = 0.08) (i.e. more benefits, less barriers) towards their children's physical activity and poor walking/cycling facilities in the neighborhood (OR = 0.70; p = 0.06) were associated with children's maintenance of active transport to leisure time destinations compared to a switch to or maintenance of passive transport. This longitudinal study can give directions for interventions promoting children's active transport during the transition to secondary school. It is necessary to promote different possibilities at primary school for children to use active transport when going to secondary school. Walking/cycling a part of the home-school trip can be a possible solution for children who will be living at non-feasible distances from secondary school. Providing safe

  6. Correlation between protein secondary structure, backbone bond angles, and side-chain orientations

    Science.gov (United States)

    Lundgren, Martin; Niemi, Antti J.

    2012-08-01

    We investigate the fine structure of the sp3 hybridized covalent bond geometry that governs the tetrahedral architecture around the central Cα carbon of a protein backbone, and for this we develop new visualization techniques to analyze high-resolution x-ray structures in the Protein Data Bank. We observe that there is a correlation between the deformations of the ideal tetrahedral symmetry and the local secondary structure of the protein. We propose a universal coarse-grained energy function to describe the ensuing side-chain geometry in terms of the Cβ carbon orientations. The energy function can model the side-chain geometry with a subatomic precision. As an example we construct the Cα-Cβ structure of HP35 chicken villin headpiece. We obtain a configuration that deviates less than 0.4 Å in root-mean-square distance from the experimental x-ray structure.

  7. Identification and expression analysis of MATE genes involved in flavonoid transport in blueberry plants.

    Science.gov (United States)

    Chen, Li; Liu, Yushan; Liu, Hongdi; Kang, Limin; Geng, Jinman; Gai, Yuzhuo; Ding, Yunlong; Sun, Haiyue; Li, Yadong

    2015-01-01

    Multidrug and toxic compound extrusion (MATE) proteins are the most recently identified family of multidrug transporters. In plants, this family is remarkably large compared to the human and bacteria counterpart, highlighting the importance of MATE proteins in this kingdom. Here 33 Unigenes annotated as MATE transporters were found in the blueberry fruit transcriptome, of which eight full-length cDNA sequences were identified and cloned. These proteins are composed of 477-517 residues, with molecular masses ~54 kDa, and theoretical isoelectric points from 5.35 to 8.41. Bioinformatics analysis predicted 10-12 putative transmembrane segments for VcMATEs, and localization to the plasma membrane without an N-terminal signal peptide. All blueberry MATE proteins shared 32.1-84.4% identity, among which VcMATE2, VcMATE3, VcMATE5, VcMATE7, VcMATE8, and VcMATE9 were more similar to the MATE-type flavonoid transporters. Phylogenetic analysis showed VcMATE2, VcMATE3, VcMATE5, VcMATE7, VcMATE8 and VcMATE9 clustered with MATE-type flavonoid transporters, indicating that they might be involved in flavonoid transport. VcMATE1 and VcMATE4 may be involved in the transport of secondary metabolites, the detoxification of xenobiotics, or the export of toxic cations. Real-time quantitative PCR demonstrated that the expression profile of the eight VcMATE genes varied spatially and temporally. Analysis of expression and anthocyanin accumulation indicated that there were some correlation between the expression profile and the accumulation of anthocyanins. These results showed VcMATEs might be involved in diverse physiological functions, and anthocyanins across the membranes might be mutually maintained by MATE-type flavonoid transporters and other mechanisms. This study will enrich the MATE-based transport mechanisms of secondary metabolite, and provide a new biotechonology strategy to develop better nutritional blueberry cultivars.

  8. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    International Nuclear Information System (INIS)

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

    2005-01-01

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

  9. Chloroplast Iron Transport Proteins - Function and Impact on Plant Physiology.

    Science.gov (United States)

    López-Millán, Ana F; Duy, Daniela; Philippar, Katrin

    2016-01-01

    Chloroplasts originated about three billion years ago by endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. During evolution chloroplasts of higher plants established as the site for photosynthesis and thus became the basis for all life dependent on oxygen and carbohydrate supply. To fulfill this task, plastid organelles are loaded with the transition metals iron, copper, and manganese, which due to their redox properties are essential for photosynthetic electron transport. In consequence, chloroplasts for example represent the iron-richest system in plant cells. However, improvement of oxygenic photosynthesis in turn required adaptation of metal transport and homeostasis since metal-catalyzed generation of reactive oxygen species (ROS) causes oxidative damage. This is most acute in chloroplasts, where radicals and transition metals are side by side and ROS-production is a usual feature of photosynthetic electron transport. Thus, on the one hand when bound by proteins, chloroplast-intrinsic metals are a prerequisite for photoautotrophic life, but on the other hand become toxic when present in their highly reactive, radical generating, free ionic forms. In consequence, transport, storage and cofactor-assembly of metal ions in plastids have to be tightly controlled and are crucial throughout plant growth and development. In the recent years, proteins for iron transport have been isolated from chloroplast envelope membranes. Here, we discuss their putative functions and impact on cellular metal homeostasis as well as photosynthetic performance and plant metabolism. We further consider the potential of proteomic analyses to identify new players in the field.

  10. Exploiting the Past and the Future in Protein Secondary Structure Prediction

    DEFF Research Database (Denmark)

    Baldi, Pierre; Brunak, Søren; Frasconi, P

    1999-01-01

    predictions based on variable ranges of dependencies. These architectures extend recurrent neural networks, introducing non-causal bidirectional dynamics to capture both upstream and downstream information. The prediction algorithm is completed by the use of mixtures of estimators that leverage evolutionary......Motivation: Predicting the secondary structure of a protein (alpha-helix, beta-sheet, coil) is an important step towards elucidating its three-dimensional structure, as well as its function. Presently, the best predictors are based on machine learning approaches, in particular neural network...

  11. SVM-PB-Pred: SVM based protein block prediction method using sequence profiles and secondary structures.

    Science.gov (United States)

    Suresh, V; Parthasarathy, S

    2014-01-01

    We developed a support vector machine based web server called SVM-PB-Pred, to predict the Protein Block for any given amino acid sequence. The input features of SVM-PB-Pred include i) sequence profiles (PSSM) and ii) actual secondary structures (SS) from DSSP method or predicted secondary structures from NPS@ and GOR4 methods. There were three combined input features PSSM+SS(DSSP), PSSM+SS(NPS@) and PSSM+SS(GOR4) used to test and train the SVM models. Similarly, four datasets RS90, DB433, LI1264 and SP1577 were used to develop the SVM models. These four SVM models developed were tested using three different benchmarking tests namely; (i) self consistency, (ii) seven fold cross validation test and (iii) independent case test. The maximum possible prediction accuracy of ~70% was observed in self consistency test for the SVM models of both LI1264 and SP1577 datasets, where PSSM+SS(DSSP) input features was used to test. The prediction accuracies were reduced to ~53% for PSSM+SS(NPS@) and ~43% for PSSM+SS(GOR4) in independent case test, for the SVM models of above two same datasets. Using our method, it is possible to predict the protein block letters for any query protein sequence with ~53% accuracy, when the SP1577 dataset and predicted secondary structure from NPS@ server were used. The SVM-PB-Pred server can be freely accessed through http://bioinfo.bdu.ac.in/~svmpbpred.

  12. Neutron secondary-particle production cross sections and their incorporation into Monte-Carlo transport codes

    International Nuclear Information System (INIS)

    Brenner, D.J.; Prael, R.E.; Little, R.C.

    1987-01-01

    Realistic simulations of the passage of fast neutrons through tissue require a large quantity of cross-sectional data. What are needed are differential (in particle type, energy and angle) cross sections. A computer code is described which produces such spectra for neutrons above ∼14 MeV incident on light nuclei such as carbon and oxygen. Comparisons have been made with experimental measurements of double-differential secondary charged-particle production on carbon and oxygen at energies from 27 to 60 MeV; they indicate that the model is adequate in this energy range. In order to utilize fully the results of these calculations, they should be incorporated into a neutron transport code. This requires defining a generalized format for describing charged-particle production, putting the calculated results in this format, interfacing the neutron transport code with these data, and charged-particle transport. The design and development of such a program is described. 13 refs., 3 figs

  13. Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Villanueva, Josep; Villegas, Virtudes; Querol, Enrique; Avilés, Francesc X; Serrano, Luis

    2002-09-01

    In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability. Copyright 2002 John Wiley & Sons, Ltd.

  14. Incorporating deep learning with convolutional neural networks and position specific scoring matrices for identifying electron transport proteins.

    Science.gov (United States)

    Le, Nguyen-Quoc-Khanh; Ho, Quang-Thai; Ou, Yu-Yen

    2017-09-05

    In several years, deep learning is a modern machine learning technique using in a variety of fields with state-of-the-art performance. Therefore, utilization of deep learning to enhance performance is also an important solution for current bioinformatics field. In this study, we try to use deep learning via convolutional neural networks and position specific scoring matrices to identify electron transport proteins, which is an important molecular function in transmembrane proteins. Our deep learning method can approach a precise model for identifying of electron transport proteins with achieved sensitivity of 80.3%, specificity of 94.4%, and accuracy of 92.3%, with MCC of 0.71 for independent dataset. The proposed technique can serve as a powerful tool for identifying electron transport proteins and can help biologists understand the function of the electron transport proteins. Moreover, this study provides a basis for further research that can enrich a field of applying deep learning in bioinformatics. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Transport of lead in secondary systems of PWR plants: laboratory and plant investigations

    International Nuclear Information System (INIS)

    Feron, D.; Rocher, A.; Nordmann, F.

    1992-01-01

    Both in France and abroad, abnormally high lead concentrations have been found in the deposits on certain steam generator tubes subject to combined inter and transgranular corrosion on the secondary side. Many potential sources of lead have been identified in PWR steam-water system, mainly at the turbine level. Tests on a loop (ORION) have shown that lead (as Pb or PbO) can transport from the condenser to the steam generator and that the contaminant mainly concentrates in flow restricted areas of steam generators

  16. Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements.

    Science.gov (United States)

    Rachel, Natalie M; Quaglia, Daniela; Lévesque, Éric; Charette, André B; Pelletier, Joelle N

    2017-11-01

    Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side-chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site-specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100-fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5-cd]indolizine-type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α-helical, β-sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q-GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG-reactive glutamines can be readily introduced into a protein domain for fluorescent labeling. © 2017 The Protein Society.

  17. Effect of electric charge on the transperitoneal transport of plasma proteins during CAPD

    NARCIS (Netherlands)

    Buis, B.; Koomen, G. C.; Imholz, A. L.; Struijk, D. G.; Reddingius, R. E.; Arisz, L.; Krediet, R. T.

    1996-01-01

    BACKGROUND: Controversy exists as to whether electric charges of plasma proteins influence their transport across the peritoneal membrane during CAPD. Fixed negative charges in the peritoneal membrane are diminished during peritonitis in rats. METHODS: Peritoneal clearances of 10 proteins and their

  18. FRACTEX: an experiment aiming at characterizing the transport properties of a secondary fault

    International Nuclear Information System (INIS)

    Wittebroodt, C.; Matray, J.M.; Dick, P.; Cabrera, J.; Barnichon, J.D.

    2012-01-01

    Document available in extended abstract form only. Because of their favourable transport and retention properties, argillaceous rocks are considered as potential host rocks for radioactive waste repositories. At the request of the French Authority of Nuclear Safety (ASN), the French Institute for Radioprotection and Nuclear Safety (IRSN) is in charge of an independent expertise of the French industrial (Andra's) project. Therefore, IRSN develops experimental research programs in such geological formations at the Tournemire Underground Research Laboratory (URL, Aveyron, France). One of the objectives of this project is to evaluate the occurrence and the driving processes controlling the radionuclide migration through an argillaceous formation similar to those studied elsewhere for nuclear waste disposal. Since undisturbed argillaceous rocks display very low values for both hydraulic conductivity (K h ) and water content (θ e ), diffusion is considered to be the main transport mechanism governing radionuclide migration through the argillite. On the other hand, in the presence of fracture in the argillaceous formation, the flux of water through this preferential pathway could dramatically accelerate the migration of the radionuclide it contains. Thus, the implementation of a radwaste disposal facility requires a precise sedimentary and structural characterization of the preselected site to guarantee the presence of a regular, homogenous and fault-free clay layer over a large area and so to determine its ability to ensure an effective radionuclide confinement. The site characterization and fracture detection can be performed using complementary approaches such as geological studies, in situ measurements and non-destructive geophysical methods. In order to evaluate both the capacity and the limit of these seismic methods to detect a secondary fault in an argillaceous media, IRSN performed a 3D HR seismic survey from the surface of its Tournemire URL. Due to the weak

  19. Plasma Membrane-Located Purine Nucleotide Transport Proteins Are Key Components for Host Exploitation by Microsporidian Intracellular Parasites

    Science.gov (United States)

    Heinz, Eva; Hacker, Christian; Dean, Paul; Mifsud, John; Goldberg, Alina V.; Williams, Tom A.; Nakjang, Sirintra; Gregory, Alison; Hirt, Robert P.; Lucocq, John M.; Kunji, Edmund R. S.; Embley, T. Martin

    2014-01-01

    Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis. PMID:25474405

  20. Plasma membrane-located purine nucleotide transport proteins are key components for host exploitation by microsporidian intracellular parasites.

    Directory of Open Access Journals (Sweden)

    Eva Heinz

    2014-12-01

    Full Text Available Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes, consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.

  1. Equilibrium and Transport Properties of Primary, Secondary and Tertiary Amines by Molecular Simulation

    International Nuclear Information System (INIS)

    Orozco, Gustavo A.; Nieto-Draghi, Carlos; Lachet, Veronique; Mackie, Allan D.

    2014-01-01

    Using molecular simulation techniques such as Monte Carlo (MC) and molecular dynamics (MD), we present several simulation results of thermodynamic and transport properties for primary, secondary and tertiary amines. These calculations are based on a recently proposed force field for amines that follows the Anisotropic United Atom approach (AUA). Different amine molecules have been studied, including n-Butylamine, di-n-Butylamine, tri-n-Butylamine and 1,4-Butanediamine for primary, secondary, tertiary and multi-functional amines respectively. For the transport properties, we have calculated the viscosity coefficients as a function of temperature using the isothermal-isobaric (NPT) ensemble. In the case of the pure components, we have investigated different thermodynamic properties using NVT Gibbs ensemble simulations such as liquid-vapor phase equilibrium diagrams, vaporization enthalpies, vapor pressures, normal boiling points, critical temperatures and critical densities. We have also calculated the excess enthalpies for water-n-Butylamine and n-heptane-n-Butylamine mixtures using Monte Carlo simulations in the NPT ensemble. In addition, we present the calculation of liquid-vapor surface tensions of n-Butylamine using a two-phase NVT simulation as well as the radial distribution functions. Finally, we have investigated the physical Henry constants of nitrous oxide (N 2 O) and nitrogen (N 2 ) in an aqueous solutions of n-Butylamine. In general, we found a good agreement between the available experimental information and our simulation results for all the studied properties, ratifying the predictive capability of the AUA force field for amines. (authors)

  2. N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

    OpenAIRE

    Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M.

    2013-01-01

    Background N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the G? subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the presen...

  3. Convective transport of highly plasma protein bound drugs facilitates direct penetration into deep tissues after topical application

    Science.gov (United States)

    Dancik, Yuri; Anissimov, Yuri G; Jepps, Owen G; Roberts, Michael S

    2012-01-01

    AIMS To relate the varying dermal, subcutaneous and muscle microdialysate concentrations found in man after topical application to the nature of the drug applied and to the underlying physiology. METHODS We developed a physiologically based pharmacokinetic model in which transport to deeper tissues was determined by tissue diffusion, blood, lymphatic and intersitial flow transport and drug properties. The model was applied to interpret published human microdialysis data, estimated in vitro dermal diffusion and protein binding affinity of drugs that have been previously applied topically in vivo and measured in deep cutaneous tissues over time. RESULTS Deeper tissue microdialysis concentrations for various drugs in vivo vary widely. Here, we show that carriage by the blood to the deeper tissues below topical application sites facilitates the transport of highly plasma protein bound drugs that penetrate the skin, leading to rapid and significant concentrations in those tissues. Hence, the fractional concentration for the highly plasma protein bound diclofenac in deeper tissues is 0.79 times that in a probe 4.5 mm below a superficial probe whereas the corresponding fractional concentration for the poorly protein bound nicotine is 0.02. Their corresponding estimated in vivo lag times for appearance of the drugs in the deeper probes were 1.1 min for diclofenac and 30 min for nicotine. CONCLUSIONS Poorly plasma protein bound drugs are mainly transported to deeper tissues after topical application by tissue diffusion whereas the transport of highly plasma protein bound drugs is additionally facilitated by convective blood, lymphatic and interstitial transport to deep tissues. PMID:21999217

  4. Phloem RNA-binding proteins as potential components of the long-distance RNA transport system.

    Directory of Open Access Journals (Sweden)

    VICENTE ePALLAS

    2013-05-01

    Full Text Available RNA-binding proteins (RBPs govern a myriad of different essential processes in eukaryotic cells. Recent evidence reveals that apart from playing critical roles in RNA metabolism and RNA transport, RBPs perform a key function in plant adaption to various environmental conditions. Long distance RNA transport occurs in land plants through the phloem, a conducting tissue that integrates the wide range of signalling pathways required to regulate plant development and response to stress processes. The macromolecules in the phloem pathway vary greatly and include defence proteins, transcription factors, chaperones acting in long distance trafficking, and RNAs (mRNAs, siRNAs and miRNAs. How these RNA molecules translocate through the phloem is not well understood, but recent evidence indicates the presence of translocatable RNA-binding proteins in the phloem, which act as potential components of long distance RNA transport system. This review updates our knowledge on the characteristics and functions of RBPs present in the phloem.

  5. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-05-18

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

  6. Impaired renal secretion of substrates for the multidrug resistance protein 2 in mutant transport-deficient (TR-) rats.

    NARCIS (Netherlands)

    Masereeuw, R.; Notenboom, S.; Smeets, P.H.E.; Wouterse, A.C.; Russel, F.G.M.

    2003-01-01

    Previous studies with mutant transport-deficient rats (TR(-)), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is

  7. Mutations that alter the transport function of the LamB protein in Escherichia coli.

    OpenAIRE

    Wandersman, C; Schwartz, M

    1982-01-01

    Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in...

  8. Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides

    DEFF Research Database (Denmark)

    Ernst, Heidi Asschenfeldt; Pham, Antony; Hald, Helle

    2009-01-01

    Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-express...

  9. A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables.

    Science.gov (United States)

    Wang, Yuanze; van Oosterwijk, Niels; Ali, Ameena M; Adawy, Alaa; Anindya, Atsarina L; Dömling, Alexander S S; Groves, Matthew R

    2017-08-24

    Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

  10. Mechanisms of EHD/RME-1 Protein Function in Endocytic Transport

    Science.gov (United States)

    Grant, Barth D.; Caplan, Steve

    2009-01-01

    The evolutionarily conserved Eps15 homology domain (EHD)/receptor-mediated endocytosis (RME)-1 family of C-terminal EH domain proteins has recently come under intense scrutiny because of its importance in intracellular membrane transport, especially with regard to the recycling of receptors from endosomes to the plasma membrane. Recent studies have shed new light on the mode by which these adenosine triphosphatases function on endosomal membranes in mammals and Caenorhabditis elegans. This review highlights our current understanding of the physiological roles of these proteins in vivo, discussing conserved features as well as emerging functional differences between individual mammalian paralogs. In addition, these findings are discussed in light of the identification of novel EHD/RME-1 protein and lipid interactions and new structural data for proteins in this family, indicating intriguing similarities to the Dynamin superfamily of large guanosine triphosphatases. PMID:18801062

  11. Pharmaceutical excipients influence the function of human uptake transporting proteins.

    Science.gov (United States)

    Engel, Anett; Oswald, Stefan; Siegmund, Werner; Keiser, Markus

    2012-09-04

    Although pharmaceutical excipients are supposed to be pharmacologically inactive, solubilizing agents like Cremophor EL have been shown to interact with cytochrome P450 (CYP)-dependent drug metabolism as well as efflux transporters such as P-glycoprotein (ABCB1) and multidrug resistance associated protein 2 (ABCC2). However, knowledge about their influence on the function of uptake transporters important in drug disposition is very limited. In this study we investigated the in vitro influence of polyethylene glycol 400 (PEG), hydroxypropyl-β-cyclodextrin (HPCD), Solutol HS 15 (SOL), and Cremophor EL (CrEL) on the organic anion transporting polypeptides (OATP) 1A2, OATP2B1, OATP1B1, and OATP1B3 and the Na(+)/taurocholate cotransporting polypeptide (NTCP). In stably transfected human embryonic kidney cells we analyzed the competition of the excipients with the uptake of bromosulfophthalein in OATP1B1, OATP1B3, OATP2B1, and NTCP, estrone-3-sulfate (E(3)S) in OATP1A2, OATP1B1, and OATP2B1, estradiol-17β-glucuronide in OATP1B3, and taurocholate (TA) in OATP1A2 and NTCP cells. SOL and CrEL were the most potent inhibitors of all transporters with the strongest effect on OATP1A2, OATP1B3, and OATP2B1 (IC(50) < 0.01%). HPCD also strongly inhibited all transport proteins but only for substrates containing a sterane-backbone. Finally, PEG seems to be a selective and potent modulator of OATP1A2 with IC(50) values of 0.05% (TA) and 0.14% (E(3)S). In conclusion, frequently used solubilizing agents were shown to interact substantially with intestinal and hepatic uptake transporters which should be considered in drug development. However, the clinical relevance of these findings needs to be evaluated in further in vivo studies.

  12. The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

    Science.gov (United States)

    Lloyd, Chelsea R; Park, Seongjin; Fei, Jingyi; Vanderpool, Carin K

    2017-06-01

    The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria. IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic

  13. Absorption of Vitamin A and Carotenoids by the Enterocyte: Focus on Transport Proteins

    Directory of Open Access Journals (Sweden)

    Emmanuelle Reboul

    2013-09-01

    Full Text Available Vitamin A deficiency is a public health problem in most developing countries, especially in children and pregnant women. It is thus a priority in health policy to improve preformed vitamin A and/or provitamin A carotenoid status in these individuals. A more accurate understanding of the molecular mechanisms of intestinal vitamin A absorption is a key step in this direction. It was long thought that β-carotene (the main provitamin A carotenoid in human diet, and thus all carotenoids, were absorbed by a passive diffusion process, and that preformed vitamin A (retinol absorption occurred via an unidentified energy-dependent transporter. The discovery of proteins able to facilitate carotenoid uptake and secretion by the enterocyte during the past decade has challenged established assumptions, and the elucidation of the mechanisms of retinol intestinal absorption is in progress. After an overview of vitamin A and carotenoid fate during gastro-duodenal digestion, our focus will be directed to the putative or identified proteins participating in the intestinal membrane and cellular transport of vitamin A and carotenoids across the enterocyte (i.e., Scavenger Receptors or Cellular Retinol Binding Proteins, among others. Further progress in the identification of the proteins involved in intestinal transport of vitamin A and carotenoids across the enterocyte is of major importance for optimizing their bioavailability.

  14. Characterization of PM2.5 and identification of transported secondary and biomass burning contribution in Seoul, Korea.

    Science.gov (United States)

    Kim, Yumi; Seo, Jihoon; Kim, Jin Young; Lee, Ji Yi; Kim, Hwajin; Kim, Bong Mann

    2018-02-01

    The chemical and seasonal characteristics of fine particulates in Seoul, Korea, were investigated based on 24-h integrated PM 2.5 measurements made over four 1-month periods in each season between October 2012 and September 2013. The four-season average concentration of PM 2.5 was 37 μg m -3 , and the major chemical components were secondary inorganic aerosol (SIA) species of sulfate, nitrate, and ammonium (49%), followed by organic matter (34%). The mass concentration and most of the chemical components of PM 2.5 showed clear seasonal variation, with a winter-high and summer-low pattern. The winter-to-summer sulfate ratio and the winter organic carbon (OC)-to-elemental carbon (EC) ratio were unusually high compared with those in previous studies. Strong correlations of both the sulfate level and the sulfur oxidation ratio with relative humidity, and between water-soluble OC (WSOC) and SIA in winter, suggest the importance of aqueous phase chemistry for secondary aerosols. A strong correlation between non-sea salt sulfate and Na + levels, a high Cl - /Na + ratio, and an unusual positive correlation between the nitrogen oxidation ratio and temperature during the winter indicate the influence of transported secondary emission sources from upwind urban areas and from China across the Yellow Sea. Despite the absence of local forest fires and the regulation of wood burning, a high levoglucosan concentration and its correlations with OC and WSOC indicate that Seoul was affected by biomass burning sources in the winter. The unusually high water-insoluble OC (WIOC)-to-EC ratio in winter implies additional transported combustion sources of WIOC. The strong correlation between WIOC and levoglucosan suggests the likely influence of transported biomass burning sources on the high WIOC/EC ratio during the winter.

  15. Nuclear transport of heat shock proteins in stressed cells

    International Nuclear Information System (INIS)

    Chughtai, Zahoor Saeed

    2001-01-01

    Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy , and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the heat shock protein hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of nutrient-depleted cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. Upon starvation, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or β-gaIactosidase to nuclei. To determine whether nuclear accumulation of Star-β-galactosidase depends on a specific nuclear carrier, I have analyzed its distribution in mutant yeast strains that carry a deletion of a single β-importin gene. With this assay I have identified Nmd5p as a β-importin required to concentrate Star-β-galactosidase in nuclei of stationary phase cells. (author)

  16. Nuclear transport of heat shock proteins in stressed cells

    Energy Technology Data Exchange (ETDEWEB)

    Chughtai, Zahoor Saeed

    2001-07-01

    Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy , and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the heat shock protein hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of nutrient-depleted cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. Upon starvation, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or {beta}-gaIactosidase to nuclei. To determine whether nuclear accumulation of Star-{beta}-galactosidase depends on a specific nuclear carrier, I have analyzed its distribution in mutant yeast strains that carry a deletion of a single {beta}-importin gene. With this assay I have identified Nmd5p as a {beta}-importin required to concentrate Star-{beta}-galactosidase in nuclei of stationary phase cells. (author)

  17. Metalloido-porins: Essentiality of Nodulin 26-like intrinsic proteins in metalloid transport.

    Science.gov (United States)

    Pommerrenig, Benjamin; Diehn, Till Arvid; Bienert, Gerd Patrick

    2015-09-01

    Metalloids are a group of physiologically important elements ranging from the essential to the highly toxic. Arsenic, antimony, germanium, and tellurium are highly toxic to plants themselves and to consumers of metalloid-contaminated plants. Boron, silicon, and selenium fulfill essential or beneficial functions in plants. However, when present at high concentrations, boron and selenium cause toxicity symptoms that are detrimental to plant fitness and yield. Consequently, all plants require efficient membrane transport systems to control the uptake and extrusion of metalloids into or out of the plant and their distribution within the plant body. Several Nodulin 26-like intrinsic proteins (NIPs) that belong to the aquaporin plant water channel protein family facilitate the diffusion of uncharged metalloid species. Genetic, physiological, and molecular evidence is that NIPs from primitive to higher plants not only transport all environmentally important metalloids, but that these proteins have a major role in the uptake, translocation, and extrusion of metalloids in plants. As most of the metalloid-permeable NIP aquaporins are impermeable or are poorly permeable to water, these NIP channel proteins should be considered as physiologically essential metalloido-porins. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  18. One and two-dimensional electrophoresis of fast axonally-transported proteins in rat nerves following acrylamide and 2,5-hexanedione exposure

    International Nuclear Information System (INIS)

    Sickles, D.W.

    1990-01-01

    Transient and repeated deficiencies in protein delivery to the axon are observed following injections of acrylamide (ACR) and 2,5-hexanedione (2,5-HD) (Sickles DW, Neurotoxicology 10: 91;103, 1989; Neurosci Abstr 14:1219, 1988). We have furthered these studies by measuring the effects of single 50 mg/kg ACR and 4 nmole/kg 2,5-HD injections on the quantity of select fast-transported proteins. Proteins were radiolabelled with 3H-leucine injections of the DRG; 1 and 2 dimensional gels were used for separation of the sciatic nerve (9-45mm distal to the ganglion) homogenates. Scintillation counting demonstrated that transport of all proteins studied were affected by both toxicants. Some variation in effect was observed; a direct correlation between molecular weight (r=0.71) and original quantity of radiolabel (r=0.80) with the percent reduction in transport was observed. Some apparent increases in transport of certain proteins were observed on the 2D gels; but this may indicate a change in the isoelectric points of these transported proteins

  19. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-01-01

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

  20. Urea transporter proteins as targets for small-molecule diuretics.

    Science.gov (United States)

    Esteva-Font, Cristina; Anderson, Marc O; Verkman, Alan S

    2015-02-01

    Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

  1. Implication of the C terminus of the Prunus necrotic ringspot virus movement protein in cell-to-cell transport and in its interaction with the coat protein.

    Science.gov (United States)

    Aparicio, Frederic; Pallás, Vicente; Sánchez-Navarro, Jesús

    2010-07-01

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for viral transport. Previous analysis with MPs of other members of the family Bromoviridae has shown that the C-terminal part of these MPs plays a critical role in the interaction with the cognate coat protein (CP) and in cell-to-cell transport. Bimolecular fluorescence complementation and overlay analysis confirm an interaction between the C-terminal 38 aa of PNRSV MP and its cognate CP. Mutational analysis of the C-terminal region of the PNRSV MP revealed that its C-terminal 38 aa are dispensable for virus transport, however, the 4 aa preceding the dispensable C terminus are necessary to target the MP to the plasmodesmata and for the functionality of the protein. The capacity of the PNRSV MP to use either a CP-dependent or a CP-independent cell-to-cell transport is discussed.

  2. Olfactory marker protein: turnover and transport in normal and regenerating neurons

    International Nuclear Information System (INIS)

    Kream, R.M.; Margolis, F.L.

    1984-01-01

    A 19,000-dalton acidic protein designated olfactory marker protein (OMP) is a cell-specific marker of mature olfactory chemosensory neurons. Intranasal irrigation of mouse olfactory epithelium with [ 35 S]methionine labeled OMP to high specific activity. Turnover and transport characteristics of 35 S-labeled OMP were compared to those of 35 S-labeled global cytosol protein in groups of young, adult, and Triton-treated adult mice. The latter contained primarily large numbers of regenerating olfactory neurons. In olfactory epithelium of young and Triton-treated mice, the specific activity of OMP was three times that of global cytosol protein, whereas in adults the two measures were equal. In all three groups, however, the rate of degradation of OMP was roughly equal to that of cytosol protein (T1/2 . 5 to 6 days). By contrast, differences in T1/2 for OMP decline in the bulb of adult, young, and Triton-treated adult mice were highly significant (T1/2's of 9.3, 6.1, and 4 to 5 days, respectively; p . 0.001). The specific activity of [35S]methionine incorporated in OMP exceeded that of the free amino acid 5-fold, indicating minimal precursor reutilization during the course of our experiments. Turnover data indicate that increased isotope incorporation into OMP in the epithelium is matched by an accelerated rate of degradation in the bulb. This may be correlated with the physiological state or developmental age of the primary neurons since in young and Triton-treated adult mice, rapidly maturing ''young'' olfactory neurons represent a larger proportion of the total population than in adults. Thus, OMP behaves as a typical, relatively slowly transported soluble protein (v . 2 to 4 mm/day, slow component b)

  3. Intracellular and transcellular transport of secretory and membrane proteins in the rat hepatocyte

    International Nuclear Information System (INIS)

    Sztul, E.S.

    1984-01-01

    The intra- and transcellular transport of hepatic secretory and membrane proteins was studied in rats in vivo using [ 3 H]fucose and [ 35 S]cyteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile and plasma were separated by SDS-PAGE and identified by fluorography. 3 H-radioactivity in Golgi fractions peaked at 10 min post injection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from the Golgi complex occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 10 min later than the bulk of content proteins. A major 80K form of Secretory Component (SC) was identified in the bile by precipitation with an anti IgA antibody. A comparative study of kinetics of transport of 35 S-labeled SC and 35 S-labeled albumin showed that albumin peaked in bile at ∼45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins which are delivered to the bile canaliculus (BC)

  4. Effect of physical training on glucose transporter protein and mRNA levels in rat adipocytes

    DEFF Research Database (Denmark)

    Stallknecht, B; Andersen, P H; Vinten, J

    1993-01-01

    Physical training increases insulin-stimulated glucose transport and the number of glucose transporters in adipocytes measured by cytochalasin B binding. In the present study we used immunoblotting to measure the abundance of two glucose transporters (GLUT-4, GLUT-1) in white adipocytes from....../or intrinsic activity). GLUT-1 protein and mRNA levels/adipocyte volume did not change with age or training....

  5. Alternative protein secretion: The Mam1 ABC transporter supports secretion of M-factor linked GFP in fission yeast

    International Nuclear Information System (INIS)

    Kjaerulff, Soren; Mueller, Sven; Jensen, Martin Roland

    2005-01-01

    To examine whether the fission yeast Mam1 ABC transporter can be used for secretion of heterologous proteins, thereby bypassing the classical secretion pathway, we have analyzed chimeric forms of the M-factor precursor. It was demonstrated that GFP can be exported when fused to both the amino-terminal prosequence from mfm1 and a CaaX motif. This secretion was dependent on the Mam1 transporter and not the classical secretion pathway. The secretion efficiency of GFP, however, was relatively low and most of the reporter protein was trapped in the vacuolar membranes. Our findings suggest that the Mam1 ABC protein is a promiscuous peptide transporter that can accommodate globular proteins of a relatively large size. Furthermore, our results help in defining the sequences required for processing and secretion of natural M-factor

  6. Secondary Structure Prediction of Protein using Resilient Back Propagation Learning Algorithm

    Directory of Open Access Journals (Sweden)

    Jyotshna Dongardive

    2015-12-01

    Full Text Available The paper proposes a neural network based approach to predict secondary structure of protein. It uses Multilayer Feed Forward Network (MLFN with resilient back propagation as the learning algorithm. Point Accepted Mutation (PAM is adopted as the encoding scheme and CB396 data set is used for the training and testing of the network. Overall accuracy of the network has been experimentally calculated with different window sizes for the sliding window scheme and by varying the number of units in the hidden layer. The best results were obtained with eleven as the window size and seven as the number of units in the hidden layer.

  7. Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism.

    Science.gov (United States)

    Joyce, Helena; McCann, Andrew; Clynes, Martin; Larkin, Annemarie

    2015-05-01

    Chemotherapy involving the use of anticancer drugs remains an important strategy in the overall management of patients with metastatic cancer. Acquisition of multidrug resistance remains a major impediment to successful chemotherapy. Drug transporters in cell membranes and intracellular drug metabolizing enzymes contribute to the resistance phenotype and determine the pharmacokinetics of anticancer drugs in the body. ATP-binding cassette (ABC) transporters mediate the transport of endogenous metabolites and xenobiotics including cytotoxic drugs out of cells. Solute carrier (SLC) transporters mediate the influx of cytotoxic drugs into cells. This review focuses on the substrate interaction of these transporters, on their biology and what role they play together with drug metabolizing enzymes in eliminating therapeutic drugs from cells. The majority of anticancer drugs are substrates for the ABC transporter and SLC transporter families. Together, these proteins have the ability to control the influx and the efflux of structurally unrelated chemotherapeutic drugs, thereby modulating the intracellular drug concentration. These interactions have important clinical implications for chemotherapy because ultimately they determine therapeutic efficacy, disease progression/relapse and the success or failure of patient treatment.

  8. Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages

    Science.gov (United States)

    Cui, Yizhi; Zhou, Yanlong; Yin, Xingfeng; Guo, Jiahui; Zhang, Gong; Wang, Tong; He, Qing-Yu

    2016-01-01

    The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit. PMID:27602764

  9. Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages.

    Science.gov (United States)

    Chen, Zhipeng; Yang, Lijuan; Cui, Yizhi; Zhou, Yanlong; Yin, Xingfeng; Guo, Jiahui; Zhang, Gong; Wang, Tong; He, Qing-Yu

    2016-10-11

    The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit.

  10. Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C’ scalar couplings (3hbJNC’)

    NARCIS (Netherlands)

    Bonvin, A.M.J.J.; Houben, K.; Guenneugues, M.N.L.; Kaptein, R.; Boelens, R.

    2001-01-01

    The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein

  11. Fiscal 2000 research report on the technology for utilizing intracellular protein transport; 2000 nendo saibonai tanpakushitsu yuso kino riyo gijutsu chosa hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Research was conducted for the establishment of 'intracellular transport engineering' for collecting eucaryotic proteins having cytotoxicity and activated proteins having escaped decomposition into an appropriate intracellular organelle by artificially manipulating the intracellular transport system for proteins in eucaryotes. In this fiscal year, element technologies and tasks necessary for the transport and activation of intracellular proteins in eucaryotes are extracted, and research was conducted on relevant patents. In a survey of the latest trends of research and development, attention was directed mainly at cells or organelles, and the details of progress in the last one year were investigated and reported, which were related to the functions of single membrane organelles excluding for double membrane bound organelles, e.g., mitochondria and chloroplast, etc., that have unique DNA (deoxyribonucleic acid) and to the molecular mechanism of transport of protein to each organelle. Furthermore, relative to each organelle, deployment of protein transport function application technology was taken up. (NEDO)

  12. N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

    Science.gov (United States)

    Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M

    2013-01-01

    N-MYC down-regulated-like (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

  13. N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

    Directory of Open Access Journals (Sweden)

    Yashwanti Mudgil

    Full Text Available N-MYC down-regulated-like (NDL proteins interact with the Gβ subunit (AGB1 of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development.Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem.NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

  14. Clinical and pH-metric characteristics of gastro-oesophageal reflux secondary to cows' milk protein allergy.

    Science.gov (United States)

    Cavataio, F; Iacono, G; Montalto, G; Soresi, M; Tumminello, M; Carroccio, A

    1996-01-01

    AIMS: The primary aim was to assess whether there were differences in symptoms, laboratory data, and oesophageal pH-metry between infants with primary gastro-oesophageal reflux and those with reflux secondary to cows' milk protein allergy (CMPA). PATIENTS AND METHODS: 96 infants (mean(SD) age 7.8(2.0) months) with either primary gastro-oesophageal reflux, reflux with CMPA, CMPA only, or none of these (controls) were studied. Symptoms, immunochemical data, and oesophageal pH were compared between the four groups and the effect of a cows' milk protein-free diet on the severity of symptoms was also assessed. RESULTS: 14 out of 47(30%) infants with gastro-oesophageal reflux had CMPA. These infants had similar symptoms to those with primary gastro-oesophageal reflux but higher concentrations of total IgE and circulating eosinophils (p gastro-oesophageal reflux secondary to CMPA and in 24 of 25 infants with CMPA only. No infants with primary gastro-oesophageal reflux and none of the controls had this pattern. A cows' milk protein-free diet was associated with a significant improvement in symptoms only in infants with gastro-oesophageal reflux with CMPA. CONCLUSION: A characteristic oesophageal pH pattern is useful in distinguishing infants with gastro-oesophageal reflux associated with CMPA. PMID:8813871

  15. Aspen SUCROSE TRANSPORTER3 Allocates Carbon into Wood Fibers1[C][W

    Science.gov (United States)

    Mahboubi, Amir; Ratke, Christine; Gorzsás, András; Kumar, Manoj; Mellerowicz, Ewa J.; Niittylä, Totte

    2013-01-01

    Wood formation in trees requires carbon import from the photosynthetic tissues. In several tree species, including Populus species, the majority of this carbon is derived from sucrose (Suc) transported in the phloem. The mechanism of radial Suc transport from phloem to developing wood is not well understood. We investigated the role of active Suc transport during secondary cell wall formation in hybrid aspen (Populus tremula × Populus tremuloides). We show that RNA interference-mediated reduction of PttSUT3 (for Suc/H+ symporter) during secondary cell wall formation in developing wood caused thinner wood fiber walls accompanied by a reduction in cellulose and an increase in lignin. Suc content in the phloem and developing wood was not significantly changed. However, after 13CO2 assimilation, the SUT3RNAi lines contained more 13C than the wild type in the Suc-containing extract of developing wood. Hence, Suc was transported into developing wood, but the Suc-derived carbon was not efficiently incorporated to wood fiber walls. A yellow fluorescent protein:PttSUT3 fusion localized to plasma membrane, suggesting that reduced Suc import into developing wood fibers was the cause of the observed cell wall phenotype. The results show the importance of active Suc transport for wood formation in a symplasmically phloem-loading tree species and identify PttSUT3 as a principal transporter for carbon delivery into secondary cell wall-forming wood fibers. PMID:24170204

  16. Molecular features contributing to virus-independent intracellular localization and dynamic behavior of the herpesvirus transport protein US9.

    Directory of Open Access Journals (Sweden)

    Manuela Pedrazzi

    Full Text Available Reaching the right destination is of vital importance for molecules, proteins, organelles, and cargoes. Thus, intracellular traffic is continuously controlled and regulated by several proteins taking part in the process. Viruses exploit this machinery, and viral proteins regulating intracellular transport have been identified as they represent valuable tools to understand and possibly direct molecules targeting and delivery. Deciphering the molecular features of viral proteins contributing to (or determining this dynamic phenotype can eventually lead to a virus-independent approach to control cellular transport and delivery. From this virus-independent perspective we looked at US9, a virion component of Herpes Simplex Virus involved in anterograde transport of the virus inside neurons of the infected host. As the natural cargo of US9-related vesicles is the virus (or its parts, defining its autonomous, virus-independent role in vesicles transport represents a prerequisite to make US9 a valuable molecular tool to study and possibly direct cellular transport. To assess the extent of this autonomous role in vesicles transport, we analyzed US9 behavior in the absence of viral infection. Based on our studies, Us9 behavior appears similar in different cell types; however, as expected, the data we obtained in neurons best represent the virus-independent properties of US9. In these primary cells, transfected US9 mostly recapitulates the behavior of US9 expressed from the viral genome. Additionally, ablation of two major phosphorylation sites (i.e. Y32Y33 and S34ES36 have no effect on protein incorporation on vesicles and on its localization on both proximal and distal regions of the cells. These results support the idea that, while US9 post-translational modification may be important to regulate cargo loading and, consequently, virion export and delivery, no additional viral functions are required for US9 role in intracellular transport.

  17. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhongshan [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom); College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Xiang, Quanju [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Department of Microbiology, College of Resource and Environment Science, Sichuan Agriculture University, Yaan 625000 (China); Zhu, Xiaofeng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Dong, Haohao [Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); He, Chuan [School of Electronics and Information, Wuhan Technical College of Communications, No. 6 Huangjiahu West Road, Hongshan District, Wuhan, Hubei 430065 (China); Wang, Haiyan; Zhang, Yizheng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Wang, Wenjian, E-mail: Wenjian166@gmail.com [Laboratory of Department of Surgery, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080 (China); Dong, Changjiang, E-mail: C.Dong@uea.ac.uk [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom)

    2014-09-26

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

  18. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    International Nuclear Information System (INIS)

    Wang, Zhongshan; Xiang, Quanju; Zhu, Xiaofeng; Dong, Haohao; He, Chuan; Wang, Haiyan; Zhang, Yizheng; Wang, Wenjian; Dong, Changjiang

    2014-01-01

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg 2+ . • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg 2+ , which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics

  19. Accurate determination of interfacial protein secondary structure by combining interfacial-sensitive amide I and amide III spectral signals.

    Science.gov (United States)

    Ye, Shuji; Li, Hongchun; Yang, Weilai; Luo, Yi

    2014-01-29

    Accurate determination of protein structures at the interface is essential to understand the nature of interfacial protein interactions, but it can only be done with a few, very limited experimental methods. Here, we demonstrate for the first time that sum frequency generation vibrational spectroscopy can unambiguously differentiate the interfacial protein secondary structures by combining surface-sensitive amide I and amide III spectral signals. This combination offers a powerful tool to directly distinguish random-coil (disordered) and α-helical structures in proteins. From a systematic study on the interactions between several antimicrobial peptides (including LKα14, mastoparan X, cecropin P1, melittin, and pardaxin) and lipid bilayers, it is found that the spectral profiles of the random-coil and α-helical structures are well separated in the amide III spectra, appearing below and above 1260 cm(-1), respectively. For the peptides with a straight backbone chain, the strength ratio for the peaks of the random-coil and α-helical structures shows a distinct linear relationship with the fraction of the disordered structure deduced from independent NMR experiments reported in the literature. It is revealed that increasing the fraction of negatively charged lipids can induce a conformational change of pardaxin from random-coil to α-helical structures. This experimental protocol can be employed for determining the interfacial protein secondary structures and dynamics in situ and in real time without extraneous labels.

  20. Effect of heat stress on protein utilization and nutrient transporters in meat-type chickens

    Science.gov (United States)

    Habashy, Walid S.; Milfort, Marie C.; Fuller, Alberta L.; Attia, Youssef A.; Rekaya, Romdhane; Aggrey, Samuel E.

    2017-12-01

    The aim of this study was to investigate the effect of heat stress (HS) on digestibility of protein and fat and the expression of nutrient transporters in broilers. Forty-eight male Cobb500 chicks were used in this study. At day 14, birds were randomly divided into two groups and kept under either constant normal temperature (25 °C) or high temperature (35 °C) in individual cages. Five birds per treatment at 1 and 12 days post-treatment were euthanized, and Pectoralis major ( P. major) and ileum were sampled for gene expression analysis. At day 33, ileal contents were collected and used for digestibility analysis. The total consumption and retention of protein and fat were significantly lower in the HS group compared to the control group. Meanwhile, the retention of crude protein per BWG was significantly higher in the HS group compared to the control group. In P. major and ileum tissues at day 1, transporters FATP1 and SGLT1 were down-regulated in the HS group. Meanwhile, FABP1 and PepT1 were down-regulated only in the ileum of the HS group. The converse was shown in P. major. The nutrient transporter FABP1 at day 12 post-HS was down-regulated in the P. major and ileum, but GLUT1 and PepT2 were down-regulated only in the ileum, and PepT1 was down-regulated only in the P. major compared with the control group. These changes in nutrient transporters suggest that high ambient temperature might change the ileum and P. major lipids, glucose, and oligopeptide transporters.

  1. X-rays diffraction characterization of corrosion products transported by secondary side of a CANDU NPP

    International Nuclear Information System (INIS)

    Dinu, A.; Tunaru, M.; Velciu, L.

    2016-01-01

    To verify the chemistry of secondary side of CANDU steam generators, Millipore filters are used to sampling from condensing extraction pump, from feed water header and blow down of steam generator. These filters retain the corrosion products as very fine particles and are used as samples in chemistry water control. X-Ray diffraction technique is the able to distinguish the different crystallographic compounds present in oxide films deposited on the Millipore filters and gives information referring to the nature of corrosion products transported in secondary side. The XRD analysis has identified the following substance in deposited layer: magnetite (Fe_3O_4), hematite (Fe_2O_3), and iron oxide hydroxide (FeOOH). By optical microscopy it was observed a brown-reddish background specific to hematite and iron oxide hydroxide, especially for filters extracted from condensing extraction pump. The black colour of crud present on filters extracted from feed water header and blow down of steam generator shows the presence of magnetite. (authors)

  2. Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima

    International Nuclear Information System (INIS)

    Ethayathulla, Abdul S.; Bessho, Yoshitaka; Shinkai, Akeo; Padmanabhan, Balasundaram; Singh, Tej P.; Kaur, Punit; Yokoyama, Shigeyuki

    2008-01-01

    The putative ABC transporter ATP-binding protein TM0222 from T. maritima was cloned, overproduced, purified and crystallized. A complete MAD diffraction data set has been collected to 2.3 Å resolution. Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6 4 22, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å 3 Da −1 , which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan

  3. Steric exclusion and protein conformation determine the localization of plasma membrane transporters

    NARCIS (Netherlands)

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-01-01

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to

  4. Ischemia - reperfusion induced changes in levels of ion transport proteins in gerbil brain

    International Nuclear Information System (INIS)

    Lehotsky, J.; Racay, P.; Kaplan, P.; Mezesova, V.; Raeymaekers, L.

    1998-01-01

    A quantitative Western blotting was used to asses the levels of ion transport proteins in gerbil brain in control and in animals after ischemic-reperfusion injury (IRI). The gene products of plasma membrane Ca 2+ pump (PMCA) were detected in the hippocampus, cerebral cortex and cerebellum. However, they showed a distinct distribution pattern. Inositol 1,4,5-triphosphate (Ins 3 ) receptor and reticular Ca 2+ pump are the most abundant in cerebellum and hippocampus. The IRI leads to a selective decrease in content of PMCA and InsP 3 receptor I isoforms. The levels of α 3 isoform of Na + pump and reticular proteins: Ca 2+ pump and calreticulin remained constant. InsP 3 receptor and organellar Ca 2+ (SERCA) are the most abundant in cerebellum and hippocampus. Ischemia and reperfusion up to 10 days leads to a signal decrease of PMCA immuno-signal. We suppose that alteration of number of ion transport proteins, can contribute to changes which participate or follow the delayed death of neurons in hippocampus. (authors)

  5. Disparate effects of p24alpha and p24delta on secretory protein transport and processing.

    Directory of Open Access Journals (Sweden)

    Jeroen R P M Strating

    Full Text Available BACKGROUND: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3, -beta(1, -gamma(3 and -delta(2 is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC. METHODOLOGY/PRINCIPAL FINDINGS: Here we find that transgene expression of p24alpha(3 or p24delta(2 specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24alpha(3 greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24delta(2-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation. CONCLUSIONS/SIGNIFICANCE: Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing.

  6. Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins.

    Science.gov (United States)

    Palmieri, Ferdinando; Agrimi, Gennaro; Blanco, Emanuela; Castegna, Alessandra; Di Noia, Maria A; Iacobazzi, Vito; Lasorsa, Francesco M; Marobbio, Carlo M T; Palmieri, Luigi; Scarcia, Pasquale; Todisco, Simona; Vozza, Angelo; Walker, John

    2006-01-01

    The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.

  7. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

    Science.gov (United States)

    Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

    2015-01-01

    The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

  8. Regulator of G protein signaling-12 modulates the dopamine transporter in ventral striatum and locomotor responses to psychostimulants.

    Science.gov (United States)

    Gross, Joshua D; Kaski, Shane W; Schroer, Adam B; Wix, Kimberley A; Siderovski, David P; Setola, Vincent

    2018-02-01

    Regulators of G protein signaling are proteins that accelerate the termination of effector stimulation after G protein-coupled receptor activation. Many regulators of G protein signaling proteins are highly expressed in the brain and therefore considered potential drug discovery targets for central nervous system pathologies; for example, here we show that RGS12 is highly expressed in microdissected mouse ventral striatum. Given a role for the ventral striatum in psychostimulant-induced locomotor activity, we tested whether Rgs12 genetic ablation affected behavioral responses to amphetamine and cocaine. RGS12 loss significantly decreased hyperlocomotion to lower doses of both amphetamine and cocaine; however, other outcomes of administration (sensitization and conditioned place preference) were unaffected, suggesting that RGS12 does not function in support of the rewarding properties of these psychostimulants. To test whether observed response changes upon RGS12 loss were caused by changes to dopamine transporter expression and/or function, we prepared crude membranes from the brains of wild-type and RGS12-null mice and measured dopamine transporter-selective [ 3 H]WIN 35428 binding, revealing an increase in dopamine transporter levels in the ventral-but not dorsal-striatum of RGS12-null mice. To address dopamine transporter function, we prepared striatal synaptosomes and measured [ 3 H]dopamine uptake. Consistent with increased [ 3 H]WIN 35428 binding, dopamine transporter-specific [ 3 H]dopamine uptake in RGS12-null ventral striatal synaptosomes was found to be increased. Decreased amphetamine-induced locomotor activity and increased [ 3 H]WIN 35428 binding were recapitulated with an independent RGS12-null mouse strain. Thus, we propose that RGS12 regulates dopamine transporter expression and function in the ventral striatum, affecting amphetamine- and cocaine-induced increases in dopamine levels that specifically elicit acute hyperlocomotor responses.

  9. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    Energy Technology Data Exchange (ETDEWEB)

    Melton, Elaina M. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Center for Cardiovascular Sciences, Albany Medical College, Albany, NY (United States); Cerny, Ronald L. [Department of Chemistry, University of Nebraska, Lincoln, NE (United States); DiRusso, Concetta C. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Black, Paul N., E-mail: pblack2@unl.edu [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States)

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  10. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    International Nuclear Information System (INIS)

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2013-01-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  11. Microvillus-Specific Protein Tyrosine Phosphatase SAP-1 Plays a Role in Regulating the Intestinal Paracellular Transport of Macromolecules.

    Science.gov (United States)

    Mori, Shingo; Kamei, Noriyasu; Murata, Yoji; Takayama, Kozo; Matozaki, Takashi; Takeda-Morishita, Mariko

    2017-09-01

    The stomach cancer-associated protein tyrosine phosphatase 1 (SAP-1) is a receptor-type protein tyrosine phosphatase that is specifically expressed on the apical membrane of the intestinal epithelium. SAP-1 is known to maintain the balance of phosphorylation of proteins together with protein kinases; however, its biological function and impact on pharmacokinetics in the intestine remain unclear. The present study, therefore, aimed at clarifying the relationship between SAP-1 and the intestinal absorption behaviors of typical transporter substrates and macromolecules. The endogenous levels of glucose and total cholesterol in the blood were similar between wild-type and SAP-1-deficient mice (Sap1 -/- ), suggesting no contribution of SAP-1 to biogenic influx. Moreover, in vitro transport study with everted ileal sacs demonstrated that there was no difference in the absorption of breast cancer resistance protein, P-glycoprotein, and peptide transporter substrates between both mice. However, absorptive clearance of macromolecular model dextrans (FD-4 and FD-10) in Sap1 -/- mice was significantly higher than that in wild-type mice, and this was confirmed by the trend of increased FD-4 absorption from colonic loops of Sap1 -/- mice. Therefore, the results of this study suggest the partial contribution of SAP-1 to the regulated transport of hydrophilic macromolecules through paracellular tight junctions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  12. Choline transport via choline transporter-like protein 1 in conditionally immortalized rat syncytiotrophoblast cell lines TR-TBT.

    Science.gov (United States)

    Lee, N-Y; Choi, H-M; Kang, Y-S

    2009-04-01

    Choline is an essential nutrient for phospholipids and acetylcholine biosynthesis in normal development of fetus. In the present study, we investigated the functional characteristics of choline transport system and inhibitory effect of cationic drugs on choline transport in rat conditionally immortalized syncytiotrophoblast cell line (TR-TBT). Choline transport was weakly Na(+) dependent and significantly influenced by extracellular pH and by membrane depolarization. The transport process of choline is saturable with Michaelis-Menten constants (K(m)) of 68microM and 130microM in TR-TBT 18d-1 and TR-TBT 18d-2 respectively. Choline uptake in the cells was inhibited by unlabeled choline and hemicholinium-3 as well as various organic cations including guanidine, amiloride and acetylcholine. However, the prototypical organic cation tetraethylammonium and cimetidine showed very little inhibitory effect of choline uptake in TR-TBT cells. RT-PCR revealed that choline transporter-like protein 1 (CTL1) and organic cation transporter 2 (OCT2) are expressed in TR-TBT cells. The transport properties of choline in TR-TBT cells were similar or identical to that of CTL1 but not OCT2. CTL1 was also detected in human placenta. In addition, several cationic drugs such as diphenhydramine and verapamil competitively inhibited choline uptake in TR-TBT 18d-1 with K(i) of 115microM and 55microM, respectively. Our results suggest that choline transport system, which has intermediate affinity and weakly Na(+) dependent, in TR-TBT seems to occur through a CTL1 and this system may have relevance with the uptake of pharmacologically important organic cation drugs.

  13. A Deep Learning Network Approach to ab initio Protein Secondary Structure Prediction.

    Science.gov (United States)

    Spencer, Matt; Eickholt, Jesse; Jianlin Cheng

    2015-01-01

    Ab initio protein secondary structure (SS) predictions are utilized to generate tertiary structure predictions, which are increasingly demanded due to the rapid discovery of proteins. Although recent developments have slightly exceeded previous methods of SS prediction, accuracy has stagnated around 80 percent and many wonder if prediction cannot be advanced beyond this ceiling. Disciplines that have traditionally employed neural networks are experimenting with novel deep learning techniques in attempts to stimulate progress. Since neural networks have historically played an important role in SS prediction, we wanted to determine whether deep learning could contribute to the advancement of this field as well. We developed an SS predictor that makes use of the position-specific scoring matrix generated by PSI-BLAST and deep learning network architectures, which we call DNSS. Graphical processing units and CUDA software optimize the deep network architecture and efficiently train the deep networks. Optimal parameters for the training process were determined, and a workflow comprising three separately trained deep networks was constructed in order to make refined predictions. This deep learning network approach was used to predict SS for a fully independent test dataset of 198 proteins, achieving a Q3 accuracy of 80.7 percent and a Sov accuracy of 74.2 percent.

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

    Science.gov (United States)

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

    2016-07-01

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

  15. Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve

    International Nuclear Information System (INIS)

    Benowitz, L.I.; Shashoua, V.E.; Yoon, M.G.

    1981-01-01

    Double labeling methods were used to identify changes in the complement of proteins synthesized in the retinal ganglion cells and transported down the optic nerve during the process of axonal regeneration. Eight to 62 days after goldfish underwent a unilateral optic nerve crush, one eye was labeled with [3H]-, the other with [14C]proline. Control and regenerating optic nerves were dissected out and homogenized together after 5 hr, a time which allowed us to examine selectively membrane-bound components which migrate in the rapid phase of axoplasmic transport. Proteins from the two sides were so-purified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the 3H and 14C incorporation patterns along the gels revealed a radical shift away from the normal labeling spectrum during regeneration, with selective changes in labeling at particular molecular weights varying over a 3-fold range. Eight days after crushing the optic nerve, the greatest increases in labeling were seen for material with apparent molecular weights of 24,000 to 27,000, 44,000, and 210,000 daltons. These peaks declined thereafter, and on days 29 to 39, the most prominent increases were at 110,000 to 140,000 daltons. These studies indicate a continuously changing pattern in the synthesis and/or degradation of proteins that are rapidly transported down the optic nerve during regeneration and point to molecular species potential significance in the establishment of the visual map upon the brain

  16. Specificity of the second binding protein of the peptide ABC-transporter (Dpp) of Lactococcus lactis IL1403

    NARCIS (Netherlands)

    Sanz, Y; Toldra, F; Renault, P; Poolman, B

    2003-01-01

    The genome sequence of Lactococcus lactis IL1403 revealed the presence of a putative peptide-binding protein-dependent ABC-transporter (Dpp). The genes for two peptide-binding proteins (dppA and dppP) precede the membrane components, which include two transmembrane protein genes (dppB and dppC) and

  17. Arabidopsis N-MYC DOWNREGULATED-LIKE1, a positive regulator of auxin transport in a G protein-mediated pathway.

    Science.gov (United States)

    Mudgil, Yashwanti; Uhrig, Joachm F; Zhou, Jiping; Temple, Brenda; Jiang, Kun; Jones, Alan M

    2009-11-01

    Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gbetagamma dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single beta (AGB1) or the two gamma (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.

  18. Protein Kinases C-Mediated Regulations of Drug Transporter Activity, Localization and Expression

    Directory of Open Access Journals (Sweden)

    Abdullah Mayati

    2017-04-01

    Full Text Available Drug transporters are now recognized as major actors in pharmacokinetics, involved notably in drug–drug interactions and drug adverse effects. Factors that govern their activity, localization and expression are therefore important to consider. In the present review, the implications of protein kinases C (PKCs in transporter regulations are summarized and discussed. Both solute carrier (SLC and ATP-binding cassette (ABC drug transporters can be regulated by PKCs-related signaling pathways. PKCs thus target activity, membrane localization and/or expression level of major influx and efflux drug transporters, in various normal and pathological types of cells and tissues, often in a PKC isoform-specific manner. PKCs are notably implicated in membrane insertion of bile acid transporters in liver and, in this way, are thought to contribute to cholestatic or choleretic effects of endogenous compounds or drugs. The exact clinical relevance of PKCs-related regulation of drug transporters in terms of drug resistance, pharmacokinetics, drug–drug interactions and drug toxicity remains however to be precisely determined. This issue is likely important to consider in the context of the development of new drugs targeting PKCs-mediated signaling pathways, for treating notably cancers, diabetes or psychiatric disorders.

  19. Prediction of arsenic and antimony transporter major intrinsic proteins from the genomes of crop plants.

    Science.gov (United States)

    Azad, Abul Kalam; Ahmed, Jahed; Alum, Md Asraful; Hasan, Md Mahbub; Ishikawa, Takahiro; Sawa, Yoshihiro

    2018-02-01

    Major intrinsic proteins (MIPs), commonly known as aquaporins, transport water and non-polar small solutes. Comparing the 3D models and the primary selectivity-related motifs (two Asn-Pro-Ala (NPA) regions, the aromatic/arginine (ar/R) selectivity filter, and Froger's positions (FPs)) of all plant MIPs that have been experimentally proven to transport arsenic (As) and antimony (Sb), some substrate-specific signature sequences (SSSS) or specificity determining sites (SDPs) have been predicted. These SSSS or SDPs were determined in 543 MIPs found in the genomes of 12 crop plants; the As and Sb transporters were predicted to be distributed in noduline-26 like intrinsic proteins (NIPs), and every plant had one or several As and Sb transporter NIPs. Phylogenetic grouping of the NIP subfamily based on the ar/R selectivity filter and FPs were linked to As and Sb transport. We further determined the group-wise substrate selectivity profiles of the NIPs in the 12 crop plants. In addition to two NPA regions, the ar/R filter, and FPs, certain amino acids especially in the pore line, loop D, and termini contribute to the functional distinctiveness of the NIP groups. Expression analysis of transcripts in different organs indicated that most of the As and Sb transporter NIPs were expressed in roots. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants.

    Science.gov (United States)

    Vigani, Gianpiero; Bashir, Khurram; Ishimaru, Yasuhiro; Lehmann, Martin; Casiraghi, Fabio Marco; Nakanishi, Hiromi; Seki, Motoaki; Geigenberger, Peter; Zocchi, Graziano; Nishizawa, Naoko K

    2016-03-01

    Iron (Fe) is an essential micronutrient for plant growth and development, and its reduced bioavailability strongly impairs mitochondrial functionality. In this work, the metabolic adjustment in the rice (Oryza sativa) mitochondrial Fe transporter knockdown mutant (mit-2) was analysed. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared with wild-type (WT) plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases accumulated strongly in mit-2 plants, indicating that alternative pathways were activated to keep the respiratory chain working. Additionally, large-scale changes in the transcriptome and metabolome were observed in mit-2 rice plants. In particular, a strong alteration (up-/down-regulation) in the expression of genes encoding enzymes of both primary and secondary metabolism was found in mutant plants. This was reflected by changes in the metabolic profiles in both roots and shoots of mit-2 plants. Significant alterations in the levels of amino acids belonging to the aspartic acid-related pathways (aspartic acid, lysine, and threonine in roots, and aspartic acid and ornithine in shoots) were found that are strictly connected to the Krebs cycle. Furthermore, some metabolites (e.g. pyruvic acid, fumaric acid, ornithine, and oligosaccharides of the raffinose family) accumulated only in the shoot of mit-2 plants, indicating possible hypoxic responses. These findings suggest that the induction of local Fe deficiency in the mitochondrial compartment of mit-2 plants differentially affects the transcript as well as the metabolic profiles in root and shoot tissues. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  1. Testosterone increases urinary calcium excretion and inhibits expression of renal calcium transport proteins.

    NARCIS (Netherlands)

    Hsu, Y.J.; Dimke, H.; Schoeber, J.P.H.; Hsu, S.C.; Lin, S.H.; Chu, P.; Hoenderop, J.G.J.; Bindels, R.J.M.

    2010-01-01

    Although gender differences in the renal handling of calcium have been reported, the overall contribution of androgens to these differences remains uncertain. We determined here whether testosterone affects active renal calcium reabsorption by regulating calcium transport proteins. Male mice had

  2. The peritoneal transport of serum proteins and neutral dextran in CAPD patients

    NARCIS (Netherlands)

    Krediet, R. T.; Koomen, G. C.; Koopman, M. G.; Hoek, F. J.; Struijk, D. G.; Boeschoten, E. W.; Arisz, L.

    1989-01-01

    The peritoneal transport of five serum proteins and intravenously-administered neutral dextran was studied in 13 CAPD patients. In all patients a study was done three hours after the administration of dextran. In nine the study was repeated after 14 hours, and in six also after 38 hours. Using gel

  3. Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

    Science.gov (United States)

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-02-05

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

  4. Fast axonal transport of 3H-leucin-labelled proteins in the unhurt and isolated optical nerve of rats

    International Nuclear Information System (INIS)

    Wagner, H.E.

    1981-01-01

    The distribution of radioactivity of amino acid molecules incorporated in protein after injection of 3 H-Leucin into the right bulb was investigated and determined along optical nerve after 1, 2, and 4 h. A slightly increased radioactivity at the point of entrance of the optical nerves into the optical duct was found. A slightly reduced axon diameter was discussed as a possible cause. The radioactivity brought into the optical nerve via the vascular system was determined by measuring the contralateral optical nerve. In relation to the axonally transported activity, it was low. The speed of the fast axonal transport is 168 mm/d. If the processes ruling the amino acids in the perikaryon are taken into consideration, the transport speed is 240 mm/d. The application of the protein synthesis prohibitor, Cycloheximide, 5 minutes after the injection of Leucinin completely prevented the appearance of axonally transported labelled proteins. When cycloheximide was administered 2 h after Leucin, a significantly loner radioactivity than in the nerve could be determined after another 2 h; i.e. the incorporation of Leucin was not completed yet after 2 h. The profile of active compounds was the same as in the control group. In other experiments, the axonal transport of labelled proteins in isolated optical nerve fibres was tested. If the separation was carried out 2 h after the injection of Leucin an extreme reduction in activity could be determined after 1 or 2 h. The continued distribution of activity after cycloheximide treatment and removal of perikarya in comparison with the control indicate the continuation of the transport, also after separation of the axon from the perikaryon. This means that, during the time of the experiment, the mechanism of the fast axonal transport functions independently of the perikaryon. (orig./MG) [de

  5. Modeling and design of light powered biomimicry micropump utilizing transporter proteins

    Science.gov (United States)

    Liu, Jin; Sze, Tsun-Kay Jackie; Dutta, Prashanta

    2014-11-01

    The creation of compact micropumps to provide steady flow has been an on-going challenge in the field of microfluidics. We present a mathematical model for a micropump utilizing Bacteriorhodopsin and sugar transporter proteins. This micropump utilizes transporter proteins as method to drive fluid flow by converting light energy into chemical potential. The fluid flow through a microchannel is simulated using the Nernst-Planck, Navier-Stokes, and continuity equations. Numerical results show that the micropump is capable of generating usable pressure. Designing parameters influencing the performance of the micropump are investigated including membrane fraction, lipid proton permeability, illumination, and channel height. The results show that there is a substantial membrane fraction region at which fluid flow is maximized. The use of lipids with low membrane proton permeability allows illumination to be used as a method to turn the pump on and off. This capability allows the micropump to be activated and shut off remotely without bulky support equipment. This modeling work provides new insights on mechanisms potentially useful for fluidic pumping in self-sustained bio-mimic microfluidic pumps. This work is supported in part by the National Science Fundation Grant CBET-1250107.

  6. Crystallization of the A-Domain of the Mannitol Transport Protein Enzyme IImtl

    NARCIS (Netherlands)

    Lammers, Leidy A.; Dijkstra, Bauke W.; Weeghel, Rob P. van; Pas, Hendri H.; Robillard, George T.

    1992-01-01

    The A-domain of the mannitol transport protein enzyme IImtl from Escherichia coli (relative molecular mass 16,300) was crystallized, both at room temperature and 4°C, from 40% polyethylene glycol 6000 (pH 8.5 to 9.0) using the hanging-drop method of vapour diffusion. The crystals have the monoclinic

  7. A finite element model for protein transport in vivo

    Directory of Open Access Journals (Sweden)

    Montas Hubert J

    2007-06-01

    Full Text Available Abstract Background Biological mass transport processes determine the behavior and function of cells, regulate interactions between synthetic agents and recipient targets, and are key elements in the design and use of biosensors. Accurately predicting the outcomes of such processes is crucial to both enhancing our understanding of how these systems function, enabling the design of effective strategies to control their function, and verifying that engineered solutions perform according to plan. Methods A Galerkin-based finite element model was developed and implemented to solve a system of two coupled partial differential equations governing biomolecule transport and reaction in live cells. The simulator was coupled, in the framework of an inverse modeling strategy, with an optimization algorithm and an experimental time series, obtained by the Fluorescence Recovery after Photobleaching (FRAP technique, to estimate biomolecule mass transport and reaction rate parameters. In the inverse algorithm, an adaptive method was implemented to calculate sensitivity matrix. A multi-criteria termination rule was developed to stop the inverse code at the solution. The applicability of the model was illustrated by simulating the mobility and binding of GFP-tagged glucocorticoid receptor in the nucleoplasm of mouse adenocarcinoma. Results The numerical simulator shows excellent agreement with the analytic solutions and experimental FRAP data. Detailed residual analysis indicates that residuals have zero mean and constant variance and are normally distributed and uncorrelated. Therefore, the necessary and sufficient criteria for least square parameter optimization, which was used in this study, were met. Conclusion The developed strategy is an efficient approach to extract as much physiochemical information from the FRAP protocol as possible. Well-posedness analysis of the inverse problem, however, indicates that the FRAP protocol provides insufficient

  8. Salinity tolerance in plants. Quantitative approach to ion transport starting from halophytes and stepping to genetic and protein engineering for manipulating ion fluxes.

    Science.gov (United States)

    Volkov, Vadim

    2015-01-01

    Ion transport is the fundamental factor determining salinity tolerance in plants. The Review starts from differences in ion transport between salt tolerant halophytes and salt-sensitive plants with an emphasis on transport of potassium and sodium via plasma membranes. The comparison provides introductory information for increasing salinity tolerance. Effects of salt stress on ion transport properties of membranes show huge opportunities for manipulating ion fluxes. Further steps require knowledge about mechanisms of ion transport and individual genes of ion transport proteins. Initially, the Review describes methods to measure ion fluxes, the independent set of techniques ensures robust and reliable basement for quantitative approach. The Review briefly summarizes current data concerning Na(+) and K(+) concentrations in cells, refers to primary thermodynamics of ion transport and gives special attention to individual ion channels and transporters. Simplified scheme of a plant cell with known transport systems at the plasma membrane and tonoplast helps to imagine the complexity of ion transport and allows choosing specific transporters for modulating ion transport. The complexity is enhanced by the influence of cell size and cell wall on ion transport. Special attention is given to ion transporters and to potassium and sodium transport by HKT, HAK, NHX, and SOS1 proteins. Comparison between non-selective cation channels and ion transporters reveals potential importance of ion transporters and the balance between the two pathways of ion transport. Further on the Review describes in detail several successful attempts to overexpress or knockout ion transporters for changing salinity tolerance. Future perspectives are questioned with more attention given to promising candidate ion channels and transporters for altered expression. Potential direction of increasing salinity tolerance by modifying ion channels and transporters using single point mutations is discussed and

  9. Salinity tolerance in plants. Quantitative approach to ion transport starting from halophytes and stepping to genetic and protein engineering for manipulating ion fluxes

    Directory of Open Access Journals (Sweden)

    Vadim eVolkov

    2015-10-01

    Full Text Available Ion transport is the fundamental factor determining salinity tolerance in plants. The Review starts from differences in ion transport between salt tolerant halophytes and salt-sensitive plants with an emphasis on transport of potassium and sodium via plasma membranes. The comparison provides introductory information for increasing salinity tolerance. Effects of salt stress on ion transport properties of membranes show huge opportunities for manipulating ion fluxes. Further steps require knowledge about mechanisms of ion transport and individual genes of ion transport proteins. Initially, the Review describes methods to measure ion fluxes, the independent set of techniques ensures robust and reliable basement for quantitative approach. The Review briefly summarises current data concerning Na+ and K+ concentrations in cells, refers to primary thermodynamics of ion transport and gives special attention to individual ion channels and transporters. Simplified scheme of a plant cell with known transport systems at the plasma membrane and tonoplast helps to imagine the complexity of ion transport and allows to choose specific transporters for modulating ion transport. The complexity is enhanced by the influence of cell size and cell wall on ion transport. Special attention is given to ion transporters and to potassium and sodium transport by HKT, HAK, NHX and SOS1 proteins. Comparison between nonselective cation channels and ion transporters reveals potential importance of ion transporters and the balance between the two pathways of ion transport. Further on the Review describes in detail several successful attempts to overexpress or knockout ion transporters for changing salinity tolerance. Future perspectives are questioned with more attention given to promising candidate ion channels and transporters for altered expression. Potential direction of increasing salinity tolerance by modifying ion channels and transporters using single point mutations is

  10. Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

    Science.gov (United States)

    Schaletzki, Yvonne; Kromrey, Marie-Luise; Bröderdorf, Susanne; Hammer, Elke; Grube, Markus; Hagen, Paul; Sucic, Sonja; Freissmuth, Michael; Völker, Uwe; Greinacher, Andreas; Rauch, Bernhard H; Kroemer, Heyo K; Jedlitschky, Gabriele

    2017-01-05

    The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

  11. Taurocholate transport by brush-border membrane vesicles from the developing rabbit ileum: Structure/function relationships

    International Nuclear Information System (INIS)

    Schwarz, S.M.; Watkins, J.B.; Ling, S.C.

    1990-01-01

    To examine the ontogenesis of bile acid transport in the rabbit ileum, brush-border membrane vesicles (12- to 20-fold purified) were prepared from 14- to 49-day-old animals. Taurocholate uptake was characterized by the emergence of secondary active, Na(+)-dependent transport at the start of weaning (21 days). Transient intravesicular accumulation (overshoot) of taurocholate occurred at 5-10 s of incubation, and the overshoot maximum increased significantly from 21 days (349.2 +/- 22.4 nmol/mg protein) to 35 days (569.0 +/- 84.3 nmol/mg protein; p less than 0.001), without further increase at maturity (49 days, not equal to 607.6 +/- 136.7 nmol/mg protein). No significant taurocholate active uptake component was noted at 14 days; however, ileal vesicles from sucklings showed carrier-mediated, Na+ D-glucose cotransport. In greater than or equal to 35-day-old rabbits, osmolarity studies at 20 s of incubation showed that only approximately 12% of [14C]taurocholate uptake was secondary to bile acid-to-membrane binding. Conversely, at 20 min, greater than 95% of radiolabel incorporation represented solute bound to the external and/or internal membrane surface. Arrhenius plots establish brush-border membrane taurocholate uptake as an intrinsic, lipid-dependent process, with a slope discontinuity between 24 and 28 degrees C, similar to the membrane lipid thermotropic transition region. Steady-state fluorescence polarization studies (1,6-diphenyl-1,3,5-hexatriene) demonstrate a temporal association between the maturation of taurocholate uptake and age-related decreases in ileal brush-border membrane fluidity. These data indicate that maturation of bile acid secondary active transport in the rabbit ileum may be regulated, at least in part, by changes in brush-border membrane lipid dynamics

  12. Signaling governed by G proteins and cAMP is crucial for growth, secondary metabolism and sexual development in Fusarium fujikuroi.

    Directory of Open Access Journals (Sweden)

    Lena Studt

    Full Text Available The plant-pathogenic fungus Fusarium fujikuroi is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs. In addition to GAs, F. fujikuroi produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA, to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s exist. In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media. Finally, sexual crosses of ffg1 mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.

  13. Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

    Science.gov (United States)

    Niles, Brad J; Clegg, Michael S; Hanna, Lynn A; Chou, Susan S; Momma, Tony Y; Hong, Heeok; Keen, Carl L

    2008-02-22

    One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

  14. Predicting protein folding pathways at the mesoscopic level based on native interactions between secondary structure elements

    Directory of Open Access Journals (Sweden)

    Sze Sing-Hoi

    2008-07-01

    Full Text Available Abstract Background Since experimental determination of protein folding pathways remains difficult, computational techniques are often used to simulate protein folding. Most current techniques to predict protein folding pathways are computationally intensive and are suitable only for small proteins. Results By assuming that the native structure of a protein is known and representing each intermediate conformation as a collection of fully folded structures in which each of them contains a set of interacting secondary structure elements, we show that it is possible to significantly reduce the conformation space while still being able to predict the most energetically favorable folding pathway of large proteins with hundreds of residues at the mesoscopic level, including the pig muscle phosphoglycerate kinase with 416 residues. The model is detailed enough to distinguish between different folding pathways of structurally very similar proteins, including the streptococcal protein G and the peptostreptococcal protein L. The model is also able to recognize the differences between the folding pathways of protein G and its two structurally similar variants NuG1 and NuG2, which are even harder to distinguish. We show that this strategy can produce accurate predictions on many other proteins with experimentally determined intermediate folding states. Conclusion Our technique is efficient enough to predict folding pathways for both large and small proteins at the mesoscopic level. Such a strategy is often the only feasible choice for large proteins. A software program implementing this strategy (SSFold is available at http://faculty.cs.tamu.edu/shsze/ssfold.

  15. A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment.

    Science.gov (United States)

    Dietz, Andrea N; Villinger, Clarissa; Becker, Stefan; Frick, Manfred; von Einem, Jens

    2018-01-01

    The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXXΦ) in pUL71. This led us to hypothesize a requirement of the YXXΦ motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXXΦ motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXXΦ mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXXΦ motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-β-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can

  16. Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

    International Nuclear Information System (INIS)

    Ballatori, Nazzareno; Hammond, Christine L.; Cunningham, Jennifer B.; Krance, Suzanne M.; Marchan, Rosemarie

    2005-01-01

    The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions

  17. A systematic review on popularity, application and characteristics of protein secondary structure prediction tools.

    Science.gov (United States)

    Kashani-Amin, Elaheh; Tabatabaei-Malazy, Ozra; Sakhteman, Amirhossein; Larijani, Bagher; Ebrahim-Habibi, Azadeh

    2018-02-27

    Prediction of proteins' secondary structure is one of the major steps in the generation of homology models. These models provide structural information which is used to design suitable ligands for potential medicinal targets. However, selecting a proper tool between multiple secondary structure prediction (SSP) options is challenging. The current study is an insight onto currently favored methods and tools, within various contexts. A systematic review was performed for a comprehensive access to recent (2013-2016) studies which used or recommended protein SSP tools. Three databases, Web of Science, PubMed and Scopus were systematically searched and 99 out of 209 studies were finally found eligible to extract data. Four categories of applications for 59 retrieved SSP tools were: (I) prediction of structural features of a given sequence, (II) evaluation of a method, (III) providing input for a new SSP method and (IV) integrating a SSP tool as a component for a program. PSIPRED was found to be the most popular tool in all four categories. JPred and tools utilizing PHD (Profile network from HeiDelberg) method occupied second and third places of popularity in categories I and II. JPred was only found in the two first categories, while PHD was present in three fields. This study provides a comprehensive insight about the recent usage of SSP tools which could be helpful for selecting a proper tool's choice. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures.

    Science.gov (United States)

    Chovancova, Eva; Pavelka, Antonin; Benes, Petr; Strnad, Ondrej; Brezovsky, Jan; Kozlikova, Barbora; Gora, Artur; Sustr, Vilem; Klvana, Martin; Medek, Petr; Biedermannova, Lada; Sochor, Jiri; Damborsky, Jiri

    2012-01-01

    Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.

  19. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures.

    Directory of Open Access Journals (Sweden)

    Eva Chovancova

    Full Text Available Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.

  20. Expression of Duodenal Iron Transporter Proteins in Diabetic Patients with and without Iron Deficiency Anemia

    Directory of Open Access Journals (Sweden)

    Efrat Broide

    2018-01-01

    Full Text Available The role of iron transport proteins in the pathogenesis of anemia in patients with diabetes mellitus (T2DM is still unclear. We investigated the expression of duodenal transporter proteins in diabetic patients with and without iron deficiency anemia (IDA. Methods. Overall, 39 patients were included: 16 with T2DM and IDA (group A, 11 with T2DM without IDA (group B, and 12 controls (group C. Duodenal mucosal expression of divalent metal transporter 1 (DMT1, ferroportin 1 (FPN, hephaestin (HEPH, and transferrin receptor 1 (TfR was evaluated by Western blotting. Chronic disease activity markers were measured as well. Results. FPN expression was increased in group A compared to group B and controls: 1.17 (0.72–1.46, 0.76 (0.53–1.04, and 0.71 (0.64–0.86, respectively (p=0.011. TfR levels were over expressed in groups A and B compared to controls: 0.39 (0.26–0.61, 0.36 (0.24–0.43, and 0.18 (0.16–0.24, respectively, (p=0.004. The three groups did not differ significantly with regard to cellular HEPH and DMT1 expression. The normal CRP and serum ferritin levels, accompanied with normal FPN among diabetic patients without IDA, do not support the association of IDA with chronic inflammatory state. Conclusion. In patients with T2DM and IDA, duodenal iron transport protein expression might be dependent on body iron stores rather than by chronic inflammation or diabetes per se.

  1. CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures

    Science.gov (United States)

    Strnad, Ondrej; Brezovsky, Jan; Kozlikova, Barbora; Gora, Artur; Sustr, Vilem; Klvana, Martin; Medek, Petr; Biedermannova, Lada; Sochor, Jiri; Damborsky, Jiri

    2012-01-01

    Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz. PMID:23093919

  2. Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy as an Analytical Method to Investigate the Secondary Structure of a Model Protein Embedded in Solid Lipid Matrices.

    Science.gov (United States)

    Zeeshan, Farrukh; Tabbassum, Misbah; Jorgensen, Lene; Medlicott, Natalie J

    2018-02-01

    Protein drugs may encounter conformational perturbations during the formulation processing of lipid-based solid dosage forms. In aqueous protein solutions, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy can investigate these conformational changes following the subtraction of spectral interference of solvent with protein amide I bands. However, in solid dosage forms, the possible spectral contribution of lipid carriers to protein amide I band may be an obstacle to determine conformational alterations. The objective of this study was to develop an ATR FT-IR spectroscopic method for the analysis of protein secondary structure embedded in solid lipid matrices. Bovine serum albumin (BSA) was chosen as a model protein, while Precirol AT05 (glycerol palmitostearate, melting point 58 ℃) was employed as the model lipid matrix. Bovine serum albumin was incorporated into lipid using physical mixing, melting and mixing, or wet granulation mixing methods. Attenuated total reflection FT-IR spectroscopy and size exclusion chromatography (SEC) were performed for the analysis of BSA secondary structure and its dissolution in aqueous media, respectively. The results showed significant interference of Precirol ATO5 with BSA amide I band which was subtracted up to 90% w/w lipid content to analyze BSA secondary structure. In addition, ATR FT-IR spectroscopy also detected thermally denatured BSA solid alone and in the presence of lipid matrix indicating its suitability for the detection of denatured protein solids in lipid matrices. Despite being in the solid state, conformational changes occurred to BSA upon incorporation into solid lipid matrices. However, the extent of these conformational alterations was found to be dependent on the mixing method employed as indicated by area overlap calculations. For instance, the melting and mixing method imparted negligible effect on BSA secondary structure, whereas the wet granulation mixing method promoted

  3. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

    Science.gov (United States)

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

    2010-02-01

    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  4. Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation.

    Science.gov (United States)

    Bera, Krishnendu; Rani, Priyanka; Kishor, Gaurav; Agarwal, Shikha; Kumar, Antresh; Singh, Durg Vijay

    2017-09-20

    ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 μs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.

  5. Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins.

    Science.gov (United States)

    Kessels, Jana Elena; Wessels, Inga; Haase, Hajo; Rink, Lothar; Uciechowski, Peter

    2016-09-01

    The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2'-deoxycytidine (AZA) increased intracellular (after 24 and 48h) and total cellular zinc levels (after 48h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48h. MT mRNA was significantly enhanced after 24h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

  6. Determination of Endosperm Protein Secondary Structure in Hard Wheat Breeding Lines using Synchrotron Infrared Microspectroscopy

    International Nuclear Information System (INIS)

    Bonwell, E.; Fisher, T.; Fritz, A.; Wetzel, D.

    2008-01-01

    One molecular aspect of mature hard wheat protein quality for breadmaking is the relative amount of endosperm protein in the a-helix form compared with that in other secondary structure forms including β-sheet. Modeling of a-helix and β-sheet absorption bands that contribute to the amide I band at 1650 cm-1 was applied to more than 1500 spectra in this study. The microscopic view of wheat endosperm is dominated by many large starch granules with protein in between. The spectrum produced from in situ microspectroscopy of this mixture is dominated by carbohydrate bands from the large starch granules that fill up the field. The high spatial resolution achievable with synchrotron infrared microspectroscopy enables revealing good in situ spectra of the protein located interstitially. Synchrotron infrared microspectroscopic mapping of 4 μm thick frozen sections of endosperm in the subaleurone region provides spectra from a large number of pixels. Pixels with protein-dominated spectra are sorted out from among adjacent pixels to minimize the starch absorption and scattering contributions. Subsequent data treatment to extract information from the amide I band requires a high signal to noise ratio. Although spectral interference of the carbohydrate band on the amide band is not a problem, the scattering produced by the large starch granules diminishes the signal to noise ratio throughout the spectrum. High density mapping was done on beamlines U2B and U10B at the National Synchrotron Light Source at Brookhaven National Laboratory, Upton, NY. Mapping with a single masked spot size of 5.5 μm diameter or confocal 5 μm x 5 μm spot size, respectively, on the two beamlines used produced spectra for new breeding lines under current consideration. Appropriate data treatment allows calculation of a numerical estimate of the a-helix population relative to other secondary protein structures from the position and shape of the amide I absorption band. Current breeding lines show a

  7. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    International Nuclear Information System (INIS)

    Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A

    2008-01-01

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property

  8. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A [Technische Universitaet Muenchen, Technische Mikrobiologie, Weihenstephaner Steig 16, 85350 Freising (Germany)], E-mail: rudi.vogel@wzw.tum.de

    2008-07-15

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  9. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    Science.gov (United States)

    Vogel, R. F.; Linke, K.; Teichert, H.; Ehrmann, M. A.

    2008-07-01

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  10. Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein

    International Nuclear Information System (INIS)

    Price, E.M.; Ratnam, M.; Rodeman, K.M.; Freisheim, J.H.

    1988-01-01

    A radioiodinated photoaffinity analogue of methotrexate, N α -(4-amino-4-deoxy-10-methyl-pteroyl)-N ε -(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degree C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degree C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets

  11. Leading survey and research report for fiscal 1999. New technology based on functions involved in intracellular protein transport; 1999 nendo saibonai tanpakushitsu yuso kino riyo gijutsu kenkyu hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    An intercellular transport technology (artificial manipulation of an intracellular protein transport system in eucaryotes) is studied for the accumulation of cytotoxic proteins, whose expression has so far been difficult, and activated proteins, which have avoided decomposition, in appropriate intracellular minute organs. The aim is to construct a system to allow foreign proteins high in productivity and quality to express themselves for production in eucaryotes. Basic surveys were conducted of the intracellular biological functions of single-membrane organelles (endoplasmic reticulum, peroxisome, vacuole/lysosome, and Golgi body), the molecular mechanism of protein transport to each organelle, and protein activation and quality control, and element technologies were extracted. For the development of novel pharmaceuticals making use of the intracellular protein transport technology, an activated protein production system was built and a search was made for transport activity impeding substances. Research tasks relative to the development of the new technologies were isolated, such as the visualization of intercellular transport. A survey was made of the market for pharmaceuticals, cosmetics, enzymes, and visualizing equipment (fluorescence microscope provided with new functions), etc. (NEDO)

  12. Characterization of a novel sialic acid transporter of the sodium solute symporter (SSS) family and in vivo comparison with known bacterial sialic acid transporters.

    Science.gov (United States)

    Severi, Emmanuele; Hosie, Arthur H F; Hawkhead, Judith A; Thomas, Gavin H

    2010-03-01

    The function of sialic acids in the biology of bacterial pathogens is reflected by the diverse range of solute transporters that can recognize these sugar acids. Here, we use an Escherichia coliDeltananT strain to characterize the function of known and proposed bacterial sialic acid transporters. We discover that the STM1128 gene from Salmonella enterica serovar Typhimurium, which encodes a member of the sodium solute symporter family, is able to restore growth on sialic acid to the DeltananT strain and is able to transport [(14)C]-sialic acid. Using the DeltananT genetic background, we performed a direct in vivo comparison of the transport properties of the STM1128 protein with those of sialic acid transporters of the major facilitator superfamily and tripartite ATP-independent periplasmic families, E. coli NanT and Haemophilus influenzae SiaPQM, respectively. This revealed that both STM1128 and SiaPQM are sodium-dependent and, unlike SiaPQM, both STM1128 and NanT are reversible secondary carriers, demonstrating qualitative functional differences in the properties of sialic acid transporters used by bacteria that colonize humans.

  13. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

    Science.gov (United States)

    McGinley, Cian; Bishop, David J

    2016-12-01

    McGinley C, Bishop DJ. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men. J Appl Physiol 121: 1290-1305, 2016. First published October 14, 2016; doi:10.1152/japplphysiol.00630.2016-This study measured the adaptive response to exercise training for each of the acid-base transport protein families, including providing isoform-specific evidence for the monocarboxylate transporter (MCT)1/4 chaperone protein basigin and for the electrogenic sodium-bicarbonate cotransporter (NBCe)1. We investigated whether 4 wk of work-matched, high-intensity interval training (HIIT), performed either just above the lactate threshold (HIITΔ20; n = 8), or close to peak aerobic power (HIITΔ90; n = 8), influenced adaptations in acid-base transport protein abundance, nonbicarbonate muscle buffer capacity (βm in vitro ), and exercise capacity in active men. Training intensity did not discriminate between adaptations for most proteins measured, with abundance of MCT1, sodium/hydrogen exchanger (NHE) 1, NBCe1, carbonic anhydrase (CA) II, and CAXIV increasing after 4 wk, whereas there was little change in CAIII and CAIV abundance. βm in vitro also did not change. However, MCT4 protein content only increased for HIITΔ20 [effect size (ES): 1.06, 90% confidence limits × / ÷ 0.77], whereas basigin protein content only increased for HIITΔ90 (ES: 1.49, × / ÷ 1.42). Repeated-sprint ability (5 × 6-s sprints; 24 s passive rest) improved similarly for both groups. Power at the lactate threshold only improved for HIITΔ20 (ES: 0.49; 90% confidence limits ± 0.38), whereas peak O 2 uptake did not change for either group. Detraining was characterized by the loss of adaptations for all of the proteins measured and for repeated-sprint ability 6 wk after removing the stimulus of HIIT. In conclusion, 4 wk of HIIT induced improvements in each of the acid-base transport protein families, but, remarkably, a 40

  14. Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

    Science.gov (United States)

    Sook, Brian R; Block, Darci R; Sumithran, Suganya; Montañez, Griselle E; Rodgers, Kenton R; Dawson, John H; Eichenbaum, Zehava; Dixon, Dabney W

    2008-02-26

    Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

  15. The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism.

    Science.gov (United States)

    Mulligan, Christopher; Fenollar-Ferrer, Cristina; Fitzgerald, Gabriel A; Vergara-Jaque, Ariela; Kaufmann, Desirée; Li, Yan; Forrest, Lucy R; Mindell, Joseph A

    2016-03-01

    Secondary transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-transporter homolog GltPh translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na(+)/succinate transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 Å and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers.

  16. Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

    Science.gov (United States)

    Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

    2016-07-01

    Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. New secondary energy systems

    International Nuclear Information System (INIS)

    Schulten, R.

    1977-01-01

    As an introduction, the FRG's energy industry situation is described, secondary energy systems to be taken into consideration are classified, and appropriate market requirements are analyzed. Dealt with is district heating, i.e. the direct transport of heat by means of circulating media, and long-distance energy, i.e. the long-distance energy transport by means of chemical conversion in closed- or open-cycle systems. In closed-cycle systems heat is transported in the form of chemical latent energy. In contrast to this, chemical energy is transported in open-cycle systems in the form of fuel gases produced by coal gasification or by thermochemical water splitting. (GG) [de

  18. Prediction of FAD binding sites in electron transport proteins according to efficient radial basis function networks and significant amino acid pairs.

    Science.gov (United States)

    Le, Nguyen-Quoc-Khanh; Ou, Yu-Yen

    2016-07-30

    Cellular respiration is a catabolic pathway for producing adenosine triphosphate (ATP) and is the most efficient process through which cells harvest energy from consumed food. When cells undergo cellular respiration, they require a pathway to keep and transfer electrons (i.e., the electron transport chain). Due to oxidation-reduction reactions, the electron transport chain produces a transmembrane proton electrochemical gradient. In case protons flow back through this membrane, this mechanical energy is converted into chemical energy by ATP synthase. The convert process is involved in producing ATP which provides energy in a lot of cellular processes. In the electron transport chain process, flavin adenine dinucleotide (FAD) is one of the most vital molecules for carrying and transferring electrons. Therefore, predicting FAD binding sites in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. We used an independent data set to evaluate the performance of the proposed method, which had an accuracy of 69.84 %. We compared the performance of the proposed method in analyzing two newly discovered electron transport protein sequences with that of the general FAD binding predictor presented by Mishra and Raghava and determined that the accuracy of the proposed method improved by 9-45 % and its Matthew's correlation coefficient was 0.14-0.5. Furthermore, the proposed method enabled reducing the number of false positives significantly and can provide useful information for biologists. We developed a method that is based on PSSM profiles and SAAPs for identifying FAD binding sites in newly discovered electron transport protein sequences. This approach achieved a significant improvement after we added SAAPs to PSSM features to analyze FAD binding proteins in the electron transport chain. The proposed method can serve as an effective tool for predicting FAD binding sites in electron

  19. The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane.

    NARCIS (Netherlands)

    Huls, M.; Brown, C.D.; Windass, A.S.; Sayer, R.; Heuvel, J.J.M.W. van den; Heemskerk, S.; Russel, F.G.M.; Masereeuw, R.

    2008-01-01

    The Breast Cancer Resistance Protein (BCRP/ABCG2) is a transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. This transporter is highly expressed in many tissues; however, in human kidney, only the mRNA was found in contrast to the mouse kidney,

  20. Metal-like transport in proteins: A new paradigm for biological electron transfer

    Science.gov (United States)

    Malvankar, Nikhil; Vargas, Madeline; Tuominen, Mark; Lovley, Derek

    2012-02-01

    Electron flow in biologically proteins generally occurs via tunneling or hopping and the possibility of electron delocalization has long been discounted. Here we report metal-like transport in protein nanofilaments, pili, of bacteria Geobacter sulfurreducens that challenges this long-standing belief [1]. Pili exhibit conductivities comparable to synthetic organic metallic nanostructures. The temperature, magnetic field and gate-voltage dependence of pili conductivity is akin to that of quasi-1D disordered metals, suggesting a metal-insulator transition. Magnetoresistance (MR) data provide evidence for quantum interference and weak localization at room temperature, as well as a temperature and field-induced crossover from negative to positive MR. Furthermore, pili can be doped with protons. Structural studies suggest the possibility of molecular pi stacking in pili, causing electron delocalization. Reducing the disorder increases the metallic nature of pili. These electronically functional proteins are a new class of electrically conductive biological proteins that can be used to generate future generation of inexpensive and environmentally-sustainable nanomaterials and nanolectronic devices such as transistors and supercapacitors. [1] Malvankar et al. Nature Nanotechnology, 6, 573-579 (2011)

  1. Functional modulation of the glutamate transporter variant GLT1b by the PDZ domain protein PICK1

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Borre, Lars; Braunstein, Thomas H

    2013-01-01

    The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulato...

  2. Lipid Raft-Based Membrane Compartmentation of a Plant Transport Protein Expressed in Saccharomyces cerevisiae

    Czech Academy of Sciences Publication Activity Database

    Grossmann, Q.; Opekarová, Miroslava; Nováková, L.; Stolz, J.; Tanner, W.

    2006-01-01

    Roč. 5, č. 6 (2006), s. 945-953 ISSN 1535-9778 R&D Projects: GA MŠk LC545 Institutional research plan: CEZ:AV0Z50200510 Keywords : saccharomyces cerevisiae * plant transport protein * hup1 Subject RIV: EE - Microbiology, Virology Impact factor: 3.707, year: 2006

  3. Monomer-dependent secondary nucleation in amyloid formation.

    Science.gov (United States)

    Linse, Sara

    2017-08-01

    Secondary nucleation of monomers on the surface of an already existing aggregate that is formed from the same kind of monomers may lead to autocatalytic amplification of a self-assembly process. Such monomer-dependent secondary nucleation occurs during the crystallization of small molecules or proteins and self-assembled materials, as well as in protein self-assembly into fibrous structures. Indications of secondary nucleation may come from analyses of kinetic experiments starting from pure monomers or monomers supplemented with a low concentration of pre-formed aggregates (seeds). More firm evidence requires additional experiments, for example those employing isotope labels to distinguish new aggregates arising from the monomer from those resulting from fragmentation of the seed. In cases of amyloid formation, secondary nucleation leads to the formation of toxic oligomers, and inhibitors of secondary nucleation may serve as starting points for therapeutic developments. Secondary nucleation displays a high degree of structural specificity and may be enhanced by mutations or screening of electrostatic repulsion.

  4. Multidrug and toxin extrusion proteins mediate cellular transport of cadmium

    International Nuclear Information System (INIS)

    Yang, Hong; Guo, Dong; Obianom, Obinna N.; Su, Tong; Polli, James E.; Shu, Yan

    2017-01-01

    Cadmium (Cd) is an environmentally prevalent toxicant posing increasing risk to human health worldwide. As compared to the extensive research in Cd tissue accumulation, little was known about the elimination of Cd, particularly its toxic form, Cd ion (Cd 2+ ). In this study, we aimed to examine whether Cd 2+ is a substrate of multidrug and toxin extrusion proteins (MATEs) that are important in renal xenobiotic elimination. HEK-293 cells overexpressing the human MATE1 (HEK-hMATE1), human MATE2-K (HEK-hMATE2-K) and mouse Mate1 (HEK-mMate1) were used to study the cellular transport and toxicity of Cd 2+ . The cells overexpressing MATEs showed a 2–4 fold increase of Cd 2+ uptake that could be blocked by the MATE inhibitor cimetidine. A saturable transport profile was observed with the Michaelis-Menten constant (K m ) of 130 ± 15.8 μM for HEK-hMATE1; 139 ± 21.3 μM for HEK-hMATE2-K; and 88.7 ± 13.5 μM for HEK-mMate1, respectively. Cd 2+ could inhibit the uptake of metformin, a substrate of MATE transporters, with the half maximal inhibitory concentration (IC 50 ) of 97.5 ± 6.0 μM, 20.2 ± 2.6 μM, and 49.9 ± 6.9 μM in HEK-hMATE1, HEK-hMATE2-K, and HEK-mMate1 cells, respectively. In addition, hMATE1 could transport preloaded Cd 2+ out of the HEK-hMATE1 cells, thus resulting in a significant decrease of Cd 2+ -induced cytotoxicity. The present study has provided the first evidence supporting that MATEs transport Cd 2+ and may function as cellular elimination machinery in Cd intoxication. - Highlights: • Cadmium is an environmentally prevalent toxicant. • Little was known regarding the elimination and detoxification of cadmium. • Cadmium ion is here demonstrated as a substrate of MATE transporters. • MATEs may function as cellular elimination machinery in cadmium detoxification.

  5. Regorafenib is transported by the organic anion transporter 1B1 and the multidrug resistance protein 2.

    Science.gov (United States)

    Ohya, Hiroki; Shibayama, Yoshihiko; Ogura, Jiro; Narumi, Katsuya; Kobayashi, Masaki; Iseki, Ken

    2015-01-01

    Regorafenib is a small molecule inhibitor of tyrosine kinases, and has been shown to improve the outcomes of patients with advanced colorectal cancer and advanced gastrointestinal stromal tumors. The transport profiles of regorafenib by various transporters were evaluated. HEK293/organic anion transporting polypeptide 1B1 (OATP1B1) cells exhibited increased drug sensitivity to regorafenib. Regorafenib inhibited the uptake of 3H-estrone sulfate by HEK293/OATP1B1 cells in a dose-dependent manner, but did not affect its elimination by P-glycoproteins. The concentration of regorafenib was significantly lower in LLC-PK1/multidrug resistance protein 2 (MRP2) cells than in LLC-PK1 cells treated with the MRP2 inhibitor, MK571. MK571 abolished the inhibitory effects of regorafenib on intracellular accumulation in LLC-PK1/MRP2 cells. The uptake of regorafenib was significantly higher in HEK293/OATP1B1 cells than in OATP1B1-mock cells. Transport kinetics values were estimated to be Km=15.9 µM and Vmax=1.24 nmol/mg/min. No significant difference was observed in regorafenib concentrations between HEK293/OATP1B3 and OATP1B3-mock cells. These results indicated that regorafenib is a substrate for MRP2 and OATP1B1, and also suggest that the substrate preference of regorafenib may implicate the pharmacokinetic profiles of regorafenib.

  6. Molecular Diagnostics of Copper-Transporting Protein Mutations Allows Early Onset Individual Therapy of Menkes Disease.

    Science.gov (United States)

    Králík, L; Flachsová, E; Hansíková, H; Saudek, V; Zeman, J; Martásek, P

    2017-01-01

    Menkes disease is a severe X-linked recessive disorder caused by a defect in the ATP7A gene, which encodes a membrane copper-transporting ATPase. Deficient activity of the ATP7A protein results in decreased intestinal absorption of copper, low copper level in serum and defective distribution of copper in tissues. The clinical symptoms are caused by decreased activities of copper-dependent enzymes and include neurodegeneration, connective tissue disorders, arterial changes and hair abnormalities. Without therapy, the disease is fatal in early infancy. Rapid diagnosis of Menkes disease and early start of copper therapy is critical for the effectiveness of treatment. We report a molecular biology-based strategy that allows early diagnosis of copper transport defects and implementation of individual therapies before the full development of pathological symptoms. Low serum copper and decreased activity of copperdependent mitochondrial cytochrome c oxidase in isolated platelets found in three patients indicated a possibility of functional defects in copper-transporting proteins, especially in the ATPA7 protein, a copper- transporting P-type ATPase. Rapid mutational screening of the ATP7A gene using high-resolution melting analysis of DNA indicated presence of mutations in the patients. Molecular investigation for mutations in the ATP7A gene revealed three nonsense mutations: c.2170C>T (p.Gln724Ter); c.3745G>T (p.Glu1249Ter); and c.3862C>T (p.Gln1288Ter). The mutation c.3745G>T (p.Glu1249Ter) has not been identified previously. Molecular analysis of the ATOX1 gene as a possible modulating factor of Menkes disease did not reveal presence of pathogenic mutations. Molecular diagnostics allowed early onset of individual therapies, adequate genetic counselling and prenatal diagnosis in the affected families.

  7. Reduction transport of corrosion products through the secondary circuit by increasing the dosage of ammonia; Reduccion del transporte de productos de corrosion a traves del circuito secundario mediante el aumento de la dosificacion de amoniaco

    Energy Technology Data Exchange (ETDEWEB)

    Espanol, J.; Boronat, M.

    2015-07-01

    Reduce transport of corrosion products through the Secondary Circuit, mainly magnetite, by increasing the dosage of ammonia in order to reduce oxides accumulation in the Steam Generators top of tube sheet where become hard sludge, as one of the TTS denting mitigation actions. (Author)

  8. Classification of a Haemophilus influenzae ABC Transporter HI1470/71 through Its Cognate Molybdate Periplasmic Binding Protein, MolA

    Energy Technology Data Exchange (ETDEWEB)

    Tirado-Lee, Leidamarie; Lee, Allen; Rees, Douglas C.; Pinkett, Heather W. (CIT); (NWU)

    2014-10-02

    molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB{sub 2}C{sub 2} (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 {angstrom} resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The {approx}100 {mu}M binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate. The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.

  9. Effects of Long-Term Protein Restriction on Meat Quality, Muscle Amino Acids, and Amino Acid Transporters in Pigs.

    Science.gov (United States)

    Yin, Jie; Li, Yuying; Zhu, Xiaotong; Han, Hui; Ren, Wenkai; Chen, Shuai; Bin, Peng; Liu, Gang; Huang, Xingguo; Fang, Rejun; Wang, Bin; Wang, Kai; Sun, Liping; Li, Tiejun; Yin, Yulong

    2017-10-25

    This study aimed to investigate the long-term effects of protein restriction from piglets to finishing pigs for 16 weeks on meat quality, muscle amino acids, and amino acid transporters. Thirty-nine piglets were randomly divided into three groups: a control (20-18-16% crude protein, CP) and two protein restricted groups (17-15-13% CP and 14-12-10% CP). The results showed that severe protein restriction (14-12-10% CP) inhibited feed intake and body weight, while moderate protein restriction (17-15-13% CP) had little effect on growth performance in pigs. Meat quality (i.e., pH, color traits, marbling, water-holding capacity, and shearing force) were tested, and the results exhibited that 14-12-10% CP treatment markedly improved muscle marbling score and increased yellowness (b*). pH value (45 min) was significantly higher in 17-15-13% CP group than that in other groups. In addition, protein restriction reduced muscle histone, arginine, valine, and isoleucine abundances and enhanced glycine and lysine concentrations compared with the control group, while the RT-PCR results showed that protein restriction downregulated amino acids transporters. Mechanistic target of rapamycin (mTOR) signaling pathway was inactivated in the moderate protein restricted group (17-15-13% CP), while severe protein restriction with dietary 14-12-10% CP markedly enhanced mTOR phosphorylation. In conclusion, long-term protein restriction affected meat quality and muscle amino acid metabolism in pigs, which might be associated with mTOR signaling pathway.

  10. A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

    Science.gov (United States)

    Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

    2017-12-01

    Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Inhibition of epithelial Na+ transport by atriopeptin, protein kinase c, and pertussis toxin

    International Nuclear Information System (INIS)

    Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A.

    1987-01-01

    The authors have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na + by atrial natriuretic peptide and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK i . Using 22 Na + fluxes, they further investigated the modulation of Na + transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na + uptake in a time- and concentration-dependent manner. Oleoyl 2-acetylglycerol and phorbol 12-myristate 13-acetate, activators of protein kinase c, inhibit Na + uptake by 93 ± 13 and 51 ± 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK i cells, inhibits 22 Na + influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na + uptake. These events may be sequentially involved in the action of atrial natriuretic peptide

  12. Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences.

    Science.gov (United States)

    Haider, Ameena J; Cox, Megan H; Jones, Natalie; Goode, Alice J; Bridge, Katherine S; Wong, Kelvin; Briggs, Deborah; Kerr, Ian D

    2015-07-17

    ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be 'rescued' by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2. © 2015 Authors.

  13. ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function

    Directory of Open Access Journals (Sweden)

    Wen-Jung Lu

    2018-03-01

    Full Text Available Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein, as well as cells lacking the outer membrane factor (OMF TolC (Tolerance to colicins. Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV, however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.

  14. Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

    Science.gov (United States)

    Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

    2009-01-01

    Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

  15. Acylated and unacylated ghrelin do not directly stimulate glucose transport in isolated rodent skeletal muscle.

    Science.gov (United States)

    Cervone, Daniel T; Dyck, David J

    2017-07-01

    Emerging evidence implicates ghrelin, a gut-derived, orexigenic hormone, as a potential mediator of insulin-responsive peripheral tissue metabolism. However, in vitro and in vivo studies assessing ghrelin's direct influence on metabolism have been controversial, particularly due to confounding factors such as the secondary rise in growth hormone (GH) after ghrelin injection. Skeletal muscle is important in the insulin-stimulated clearance of glucose, and ghrelin's exponential rise prior to a meal could potentially facilitate this. This study was aimed at elucidating any direct stimulatory action that ghrelin may have on glucose transport and insulin signaling in isolated rat skeletal muscle, in the absence of confounding secondary factors. Oxidative soleus and glycolytic extensor digitorum longus skeletal muscles were isolated from male Sprague Dawley rats in the fed state and incubated with various concentrations of acylated and unacylated ghrelin in the presence or absence of insulin. Ghrelin did not stimulate glucose transport in either muscle type, with or without insulin. Moreover, GH had no acute, direct stimulatory effect on either basal or insulin-stimulated muscle glucose transport. In agreement with the lack of observed effect on glucose transport, ghrelin and GH also had no stimulatory effect on Ser 473 AKT or Thr 172 AMPK phosphorylation, two key signaling proteins involved in glucose transport. Furthermore, to our knowledge, we are among the first to show that ghrelin can act independent of its receptor and cause an increase in calmodulin-dependent protein kinase 2 (CaMKII) phosphorylation in glycolytic muscle, although this was not associated with an increase in glucose transport. We conclude that both acylated and unacylated ghrelin have no direct, acute influence on skeletal muscle glucose transport. Furthermore, the immediate rise in GH in response to ghrelin also does not appear to directly stimulate glucose transport in muscle. © 2017 The

  16. A possible origin of viscosity in Keplerian accretion disks due to secondary perturbation: Turbulent transport without magnetic fields

    International Nuclear Information System (INIS)

    Mukhopadhyay, Banibrata; Saha, Kanak

    2011-01-01

    The origin of hydrodynamic turbulence in rotating shear flow is a long standing puzzle. Resolving it is especially important in astrophysics when the flow's angular momentum profile is Keplerian which forms an accretion disk having negligible molecular viscosity. Hence, any viscosity in such systems must be due to turbulence, arguably governed by magnetorotational instability, especially when temperature T > or approx. 10 5 . However, such disks around quiescent cataclysmic variables, protoplanetary and star-forming disks, and the outer regions of disks in active galactic nuclei are practically neutral in charge because of their low temperature, and thus are not expected to be coupled with magnetic fields enough to generate any transport due to the magnetorotational instability. This flow is similar to plane Couette flow including the Coriolis force, at least locally. What drives their turbulence and then transport, when such flows do not exhibit any unstable mode under linear hydrodynamic perturbation? We demonstrate that the three-dimensional secondary disturbance to the primarily perturbed flow that triggers elliptical instability may generate significant turbulent viscosity in the range 0.0001 ∼ t ∼< 0.1, which can explain transport in accretion flows.

  17. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

    KAUST Repository

    Weng, Jingwei; Gu, Shuo; Gao, Xin; Huang, Xuhui; Wang, Wenning

    2017-01-01

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

  18. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

    KAUST Repository

    Weng, Jingwei

    2017-02-23

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

  19. Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

    Science.gov (United States)

    Rehan, Shahid; Jaakola, Veli-Pekka

    2015-10-01

    Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Multi-functional roles for the polypeptide transport associated domains of Toc75 in chloroplast protein import

    Science.gov (United States)

    Paila, Yamuna D; Richardson, Lynn GL; Inoue, Hitoshi; Parks, Elizabeth S; McMahon, James; Inoue, Kentaro; Schnell, Danny J

    2016-01-01

    Toc75 plays a central role in chloroplast biogenesis in plants as the membrane channel of the protein import translocon at the outer envelope of chloroplasts (TOC). Toc75 is a member of the Omp85 family of bacterial and organellar membrane insertases, characterized by N-terminal POTRA (polypeptide-transport associated) domains and C-terminal membrane-integrated β-barrels. We demonstrate that the Toc75 POTRA domains are essential for protein import and contribute to interactions with TOC receptors, thereby coupling preprotein recognition at the chloroplast surface with membrane translocation. The POTRA domains also interact with preproteins and mediate the recruitment of molecular chaperones in the intermembrane space to facilitate membrane transport. Our studies are consistent with the multi-functional roles of POTRA domains observed in other Omp85 family members and demonstrate that the domains of Toc75 have evolved unique properties specific to the acquisition of protein import during endosymbiotic evolution of the TOC system in plastids. DOI: http://dx.doi.org/10.7554/eLife.12631.001 PMID:26999824

  1. Expression of transcellular and paracellular calcium and magnesium transport proteins in renal and intestinal epithelia during lactation.

    Science.gov (United States)

    Beggs, Megan R; Appel, Ida; Svenningsen, Per; Skjødt, Karsten; Alexander, R Todd; Dimke, Henrik

    2017-09-01

    Significant alterations in maternal calcium (Ca 2+ ) and magnesium (Mg 2+ ) balance occur during lactation. Ca 2+ is the primary divalent cation mobilized into breast milk by demineralization of the skeleton and alterations in intestinal and renal Ca 2+ transport. Mg 2+ is also concentrated in breast milk, but the underlying mechanisms are not well understood. To determine the molecular alterations in Ca 2+ and Mg 2+ transport in the intestine and kidney during lactation, three groups of female mice consisting of either nonpregnant controls, lactating mice, or mice undergoing involution were examined. The fractional excretion of Ca 2+ , but not Mg 2+ , rose significantly during lactation. Renal 1-α hydroxylase and 24-OHase mRNA levels increased markedly, as did plasma 1,25 dihydroxyvitamin D levels. This was accompanied by significant increases in intestinal expression of Trpv6 and S100g in lactating mice. However, no alterations in the expression of cation-permeable claudin-2, claudin-12, or claudins-15 were found in the intestine. In the kidney, increased expression of Trpv5 and Calb1 was observed during lactation, while no changes in claudins involved in Ca 2+ and Mg 2+ transport (claudin-2, claudin-14, claudin-16, or claudin-19) were found. Consistent with the mRNA expression, expression of both calbindin-D 28K and transient receptor potential vanilloid 5 (TRPV5) proteins increased. Colonic Trpm6 expression increased during lactation, while renal Trpm6 remained unaltered. In conclusion, proteins involved in transcellular Ca 2+ and Mg 2+ transport pathways increase during lactation, while expression of paracellular transport proteins remained unchanged. Increased fractional Ca 2+ excretion can be explained by vitamin D-dependent intestinal hyperabsorption and bone demineralization, despite enhanced transcellular Ca 2+ uptake by the kidney. Copyright © 2017 the American Physiological Society.

  2. Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

    Directory of Open Access Journals (Sweden)

    Wüstner Daniel

    2012-11-01

    Full Text Available Abstract Background Fluorescence loss in photobleaching (FLIP is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp function is fitted to fluorescence loss (FL inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP, we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ disease proteins like mutant huntingtin (mtHtt can form large aggregates called inclusion bodies (IB’s. The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and

  3. Determination of the secondary structure content of proteins in aqueous solutions from their amide I and amide II infrared bands. Comparison between classical and partial least-squares methods

    International Nuclear Information System (INIS)

    Dousseau, F.; Pezolet, M.

    1990-01-01

    A method for estimating protein secondary structure from infrared spectra has been developed. The infrared spectra of H 2 O solutions of 13 proteins of known crystal structure have been recorded and corrected for the spectral contribution of water in the amide I and II region by using the algorithm of Dousseau et al. This calibration set of proteins has been analyzed by using either a classical least-squares (CLS) method or the partial least-squares (PLS) method. The pure-structure spectra calculated by the classical least-squares method are in good agreement with spectra of poly(L-lysine) in the α-helix, β-sheet, and undefined conformations. The results show that the best agreement between the secondary structure determined by X-ray crystal-lography and that predicted by infrared spectroscopy is obtained when both the amide I and II bands are used to generate the calibration set, when the PLS method is used, and when it is assumed that the secondary structure of proteins is composed of only four types of structure: ordered and disordered α-helices, β-sheet, and undefined conformation. Attempts to include turns in the secondary structure estimation have led to a loss of accuracy. The spectra of the calibration proteins were also recorded in 2 H 2 O solution. After correction for the contribution of the combination band of 2 H 2 O in the amide I' band region, the spectra were analyzed with PLS, but the results were not as good as for the spectra obtained in H 2 O, especially for the α-helical conformation

  4. Colloid transport in saturated porous media: Elimination of attachment efficiency in a new colloid transport model

    Science.gov (United States)

    Landkamer, Lee L.; Harvey, Ronald W.; Scheibe, Timothy D.; Ryan, Joseph N.

    2013-01-01

    A colloid transport model is introduced that is conceptually simple yet captures the essential features of colloid transport and retention in saturated porous media when colloid retention is dominated by the secondary minimum because an electrostatic barrier inhibits substantial deposition in the primary minimum. This model is based on conventional colloid filtration theory (CFT) but eliminates the empirical concept of attachment efficiency. The colloid deposition rate is computed directly from CFT by assuming all predicted interceptions of colloids by collectors result in at least temporary deposition in the secondary minimum. Also, a new paradigm for colloid re-entrainment based on colloid population heterogeneity is introduced. To accomplish this, the initial colloid population is divided into two fractions. One fraction, by virtue of physiochemical characteristics (e.g., size and charge), will always be re-entrained after capture in a secondary minimum. The remaining fraction of colloids, again as a result of physiochemical characteristics, will be retained “irreversibly” when captured by a secondary minimum. Assuming the dispersion coefficient can be estimated from tracer behavior, this model has only two fitting parameters: (1) the fraction of the initial colloid population that will be retained “irreversibly” upon interception by a secondary minimum, and (2) the rate at which reversibly retained colloids leave the secondary minimum. These two parameters were correlated to the depth of the Derjaguin-Landau-Verwey-Overbeek (DLVO) secondary energy minimum and pore-water velocity, two physical forces that influence colloid transport. Given this correlation, the model serves as a heuristic tool for exploring the influence of physical parameters such as surface potential and fluid velocity on colloid transport.

  5. Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

    Science.gov (United States)

    Wang, Dongdong; Tosevska, Anela; Heiß, Elke H; Ladurner, Angela; Mölzer, Christine; Wallner, Marlies; Bulmer, Andrew; Wagner, Karl-Heinz; Dirsch, Verena M; Atanasov, Atanas G

    2017-04-28

    Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 μmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease. © 2017 The Authors. Published on

  6. Protein τ-mediated effects on rat hippocampal choline transporters CHT1 and τ-amyloid β interactions

    Czech Academy of Sciences Publication Activity Database

    Krištofíková, Z.; Řípová, D.; Hegnerová, Kateřina; Šírová, J.; Homola, Jiří

    2013-01-01

    Roč. 38, č. 9 (2013), s. 1949-1959 ISSN 0364-3190 Institutional support: RVO:67985882 Keywords : Tau protein * Amyloid β peptide * Choline transporter Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 2.551, year: 2013

  7. The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport.

    Science.gov (United States)

    Ochiai, Yusuke; Uchida, Yasuo; Ohtsuki, Sumio; Tachikawa, Masanori; Aizawa, Sanshiro; Terasaki, Tetsuya

    2017-05-01

    We purposed to clarify the contribution of fatty acid transport protein 1 (FATP1/SLC 27A1) to the supply of docosahexaenoic acid (DHA) to the brain across the blood-brain barrier in this study. Transport experiments showed that the uptake rate of [ 14 C]-DHA in human FATP1-expressing HEK293 cells was significantly greater than that in empty vector-transfected (mock) HEK293 cells. The steady-state intracellular DHA concentration was nearly 2-fold smaller in FATP1-expressing than in mock cells, suggesting that FATP1 works as not only an influx, but also an efflux transporter for DHA. [ 14 C]-DHA uptake by a human cerebral microvascular endothelial cell line (hCMEC/D3) increased in a time-dependent manner, and was inhibited by unlabeled DHA and a known FATP1 substrate, oleic acid. Knock-down of FATP1 in hCMEC/D3 cells with specific siRNA showed that FATP1-mediated uptake accounts for 59.2-73.0% of total [ 14 C]-DHA uptake by the cells. Insulin treatment for 30 min induced translocation of FATP1 protein to the plasma membrane in hCMEC/D3 cells and enhanced [ 14 C]-DHA uptake. Immunohistochemical analysis of mouse brain sections showed that FATP1 protein is preferentially localized at the basal membrane of brain microvessel endothelial cells. We found that two neuroprotective substances, taurine and biotin, in addition to DHA, undergo FATP1-mediated efflux. Overall, our results suggest that FATP1 localized at the basal membrane of brain microvessels contributes to the transport of DHA, taurine and biotin into the brain, and insulin rapidly increases DHA supply to the brain by promoting translocation of FATP1 to the membrane. Read the Editorial Comment for this article on page 324. © 2016 International Society for Neurochemistry.

  8. Deciphering the shape and deformation of secondary structures through local conformation analysis

    Directory of Open Access Journals (Sweden)

    Camproux Anne-Claude

    2011-02-01

    Full Text Available Abstract Background Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Results Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. Conclusion The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons.

  9. Deciphering the shape and deformation of secondary structures through local conformation analysis.

    Science.gov (United States)

    Baussand, Julie; Camproux, Anne-Claude

    2011-02-01

    Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons.

  10. Multidrug and toxin extrusion proteins mediate cellular transport of cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hong; Guo, Dong; Obianom, Obinna N. [Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, MD (United States); Su, Tong [Department of Oral Maxillofacial Surgery, the First Affiliated Hospital, Xiangya Medical School, Central South University, Hunan 410007 (China); Polli, James E. [Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, MD (United States); Shu, Yan, E-mail: yshu@rx.umaryland.edu [Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, MD (United States)

    2017-01-01

    Cadmium (Cd) is an environmentally prevalent toxicant posing increasing risk to human health worldwide. As compared to the extensive research in Cd tissue accumulation, little was known about the elimination of Cd, particularly its toxic form, Cd ion (Cd{sup 2+}). In this study, we aimed to examine whether Cd{sup 2+} is a substrate of multidrug and toxin extrusion proteins (MATEs) that are important in renal xenobiotic elimination. HEK-293 cells overexpressing the human MATE1 (HEK-hMATE1), human MATE2-K (HEK-hMATE2-K) and mouse Mate1 (HEK-mMate1) were used to study the cellular transport and toxicity of Cd{sup 2+}. The cells overexpressing MATEs showed a 2–4 fold increase of Cd{sup 2+} uptake that could be blocked by the MATE inhibitor cimetidine. A saturable transport profile was observed with the Michaelis-Menten constant (K{sub m}) of 130 ± 15.8 μM for HEK-hMATE1; 139 ± 21.3 μM for HEK-hMATE2-K; and 88.7 ± 13.5 μM for HEK-mMate1, respectively. Cd{sup 2+} could inhibit the uptake of metformin, a substrate of MATE transporters, with the half maximal inhibitory concentration (IC{sub 50}) of 97.5 ± 6.0 μM, 20.2 ± 2.6 μM, and 49.9 ± 6.9 μM in HEK-hMATE1, HEK-hMATE2-K, and HEK-mMate1 cells, respectively. In addition, hMATE1 could transport preloaded Cd{sup 2+} out of the HEK-hMATE1 cells, thus resulting in a significant decrease of Cd{sup 2+}-induced cytotoxicity. The present study has provided the first evidence supporting that MATEs transport Cd{sup 2+} and may function as cellular elimination machinery in Cd intoxication. - Highlights: • Cadmium is an environmentally prevalent toxicant. • Little was known regarding the elimination and detoxification of cadmium. • Cadmium ion is here demonstrated as a substrate of MATE transporters. • MATEs may function as cellular elimination machinery in cadmium detoxification.

  11. The TULIP superfamily of eukaryotic lipid-binding proteins as a mediator of lipid sensing and transport.

    Science.gov (United States)

    Alva, Vikram; Lupas, Andrei N

    2016-08-01

    The tubular lipid-binding (TULIP) superfamily has emerged in recent years as a major mediator of lipid sensing and transport in eukaryotes. It currently encompasses three protein families, SMP-like, BPI-like, and Takeout-like, which share a common fold. This fold consists of a long helix wrapped in a highly curved anti-parallel β-sheet, enclosing a central, lipophilic cavity. The SMP-like proteins, which include subunits of the ERMES complex and the extended synaptotagmins (E-Syts), appear to be mainly located at membrane contacts sites (MCSs) between organelles, mediating inter-organelle lipid exchange. The BPI-like proteins, which include the bactericidal/permeability-increasing protein (BPI), the LPS (lipopolysaccharide)-binding protein (LBP), the cholesteryl ester transfer protein (CETP), and the phospholipid transfer protein (PLTP), are either involved in innate immunity against bacteria through their ability to sense lipopolysaccharides, as is the case for BPI and LBP, or in lipid exchange between lipoprotein particles, as is the case for CETP and PLTP. The Takeout-like proteins, which are comprised of insect juvenile hormone-binding proteins and arthropod allergens, transport, where known, lipid hormones to target tissues during insect development. In all cases, the activity of these proteins is underpinned by their ability to bind large, hydrophobic ligands in their central cavity and segregate them away from the aqueous environment. Furthermore, where they are involved in lipid exchange, recent structural studies have highlighted their ability to establish lipophilic, tubular channels, either between organelles in the case of SMP domains or between lipoprotein particles in the case of CETP. Here, we review the current knowledge on the structure, versatile functions, and evolution of the TULIP superfamily. We propose a deep evolutionary split in this superfamily, predating the Last Eukaryotic Common Ancestor, between the SMP-like proteins, which act on

  12. Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP 4 and breast cancer resistance protein (BCRP

    Directory of Open Access Journals (Sweden)

    Rhiannon N. Hardwick

    2016-12-01

    Full Text Available Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI and aurora (AKI kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC transporters such as P-glycoprotein and breast cancer resistance protein (BCRP, which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs, remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC50 values were estimated by determining TKI/AKI inhibition of MRP4-mediated [3H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [3H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (Css to the IC50 for each compound was calculated with Css/IC50 ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of

  13. OpenDMAP: An open source, ontology-driven concept analysis engine, with applications to capturing knowledge regarding protein transport, protein interactions and cell-type-specific gene expression

    Directory of Open Access Journals (Sweden)

    Johnson Helen L

    2008-01-01

    Full Text Available Abstract Background Information extraction (IE efforts are widely acknowledged to be important in harnessing the rapid advance of biomedical knowledge, particularly in areas where important factual information is published in a diverse literature. Here we report on the design, implementation and several evaluations of OpenDMAP, an ontology-driven, integrated concept analysis system. It significantly advances the state of the art in information extraction by leveraging knowledge in ontological resources, integrating diverse text processing applications, and using an expanded pattern language that allows the mixing of syntactic and semantic elements and variable ordering. Results OpenDMAP information extraction systems were produced for extracting protein transport assertions (transport, protein-protein interaction assertions (interaction and assertions that a gene is expressed in a cell type (expression. Evaluations were performed on each system, resulting in F-scores ranging from .26 – .72 (precision .39 – .85, recall .16 – .85. Additionally, each of these systems was run over all abstracts in MEDLINE, producing a total of 72,460 transport instances, 265,795 interaction instances and 176,153 expression instances. Conclusion OpenDMAP advances the performance standards for extracting protein-protein interaction predications from the full texts of biomedical research articles. Furthermore, this level of performance appears to generalize to other information extraction tasks, including extracting information about predicates of more than two arguments. The output of the information extraction system is always constructed from elements of an ontology, ensuring that the knowledge representation is grounded with respect to a carefully constructed model of reality. The results of these efforts can be used to increase the efficiency of manual curation efforts and to provide additional features in systems that integrate multiple sources for

  14. Staphylococcus aureus manganese transport protein C (MntC is an extracellular matrix- and plasminogen-binding protein.

    Directory of Open Access Journals (Sweden)

    Natália Salazar

    Full Text Available Infections caused by Staphylococcus aureus--particularly nosocomial infections--represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM. Manganese transport protein C (MntC, a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA. The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

  15. Modeling and dynamics of the inward-facing state of a Na+/Cl- dependent neurotransmitter transporter homologue.

    Directory of Open Access Journals (Sweden)

    Saher Afshan Shaikh

    2010-08-01

    Full Text Available The leucine transporter (LeuT has recently commanded exceptional attention due mainly to two distinctions; it provides the only crystal structures available for a protein homologous to the pharmacologically relevant neurotransmitter: sodium symporters (NSS, and, it exhibits a hallmark 5-TM inverted repeat ("LeuT-fold", a fold recently discovered to also exist in several secondary transporter families, underscoring its general role in transporter function. Constructing the transport cycle of "LeuT-fold" transporters requires detailed structural and dynamic descriptions of the outward-facing (OF and inward-facing (IF states, as well as the intermediate states. To this end, we have modeled the structurally unknown IF state of LeuT, based on the known crystal structures of the OF state of LeuT and the IF state of vSGLT, a "LeuT-fold" transporter. The detailed methodology developed for the study combines structure-based alignment, threading, targeted MD and equilibrium MD, and can be applied to other proteins. The resulting IF-state models maintain the secondary structural features of LeuT. Water penetration and solvent accessibility calculations show that TM1, TM3, TM6 and TM8 line the substrate binding/unbinding pathway with TM10 and its pseudosymmetric partner, TM5, participating in the extracellular and intracellular halves of the lumen, respectively. We report conformational hotspots where notable changes in interactions occur between the IF and OF states. We observe Na2 exiting the LeuT-substrate- complex in the IF state, mainly due to TM1 bending. Inducing a transition in only one of the two pseudosymmetric domains, while allowing the second to respond dynamically, is found to be sufficient to induce the formation of the IF state. We also propose that TM2 and TM7 may be facilitators of TM1 and TM6 motion. Thus, this study not only presents a novel modeling methodology applied to obtain the IF state of LeuT, but also describes structural

  16. Electronic transport in single-helical protein molecules: Effects of multiple charge conduction pathways and helical symmetry

    Energy Technology Data Exchange (ETDEWEB)

    Kundu, Sourav, E-mail: sourav.kunduphy@gmail.com; Karmakar, S.N.

    2016-07-15

    We propose a tight-binding model to investigate electronic transport properties of single helical protein molecules incorporating both the helical symmetry and the possibility of multiple charge transfer pathways. Our study reveals that due to existence of both the multiple charge transfer pathways and helical symmetry, the transport properties are quite rigid under influence of environmental fluctuations which indicates that these biomolecules can serve as better alternatives in nanoelectronic devices than its other biological counterparts e.g., single-stranded DNA.

  17. Phylogenetic profiles of all membrane transport proteins of the malaria parasite highlight new drug targets

    Directory of Open Access Journals (Sweden)

    January Weiner 3rd

    2016-08-01

    Full Text Available In order to combat the on-going malaria epidemic, discovery of new drug targets remains vital. Proteins that are essential to survival and specific to malaria parasites are key candidates. To survive within host cells, the parasites need to acquire nutrients and dispose of waste products across multiple membranes. Additionally, like all eukaryotes, they must redistribute ions and organic molecules between their various internal membrane bound compartments. Membrane transport proteins mediate all of these processes and are considered important mediators of drug resistance as well as drug targets in their own right. Recently, using advanced experimental genetic approaches and streamlined life cycle profiling, we generated a large collection of Plasmodium berghei gene deletion mutants and assigned essential gene functions, highlighting potential targets for prophylactic, therapeutic, and transmission-blocking anti-malarial drugs. Here, we present a comprehensive orthology assignment of all Plasmodium falciparum putative membrane transport proteins and provide a detailed overview of the associated essential gene functions obtained through experimental genetics studies in human and murine model parasites. Furthermore, we discuss the phylogeny of selected potential drug targets identified in our functional screen. We extensively discuss the results in the context of the functional assignments obtained using gene targeting available to date.

  18. Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane.

    Science.gov (United States)

    De Gois, Stéphanie; Slama, Patrick; Pietrancosta, Nicolas; Erdozain, Amaia M; Louis, Franck; Bouvrais-Veret, Caroline; Daviet, Laurent; Giros, Bruno

    2015-07-17

    Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Yeast two-hybrid screens imply involvement of Fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

    Science.gov (United States)

    Reuter, Tanja Y; Medhurst, Annette L; Waisfisz, Quinten; Zhi, Yu; Herterich, Sabine; Hoehn, Holger; Gross, Hans J; Joenje, Hans; Hoatlin, Maureen E; Mathew, Christopher G; Huber, Pia A J

    2003-10-01

    Mutations in one of at least eight different genes cause bone marrow failure, chromosome instability, and predisposition to cancer associated with the rare genetic syndrome Fanconi anemia (FA). The cloning of seven genes has provided the tools to study the molecular pathway disrupted in Fanconi anemia patients. The structure of the genes and their gene products provided few clues to their functional role. We report here the use of 3 FA proteins, FANCA, FANCC, and FANCG, as "baits" in the hunt for interactors to obtain clues for FA protein functions. Using five different human cDNA libraries we screened 36.5x10(6) clones with the technique of the yeast two-hybrid system. We identified 69 proteins which have not previously been linked to the FA pathway as direct interactors of FANCA, FANCC, or FANCG. Most of these proteins are associated with four functional classes including transcription regulation (21 proteins), signaling (13 proteins), oxidative metabolism (10 proteins), and intracellular transport (11 proteins). Interaction with 6 proteins, DAXX, Ran, IkappaBgamma, USP14, and the previously reported SNX5 and FAZF, was additionally confirmed by coimmunoprecipitation and/or colocalization studies. Taken together, our data strongly support the hypothesis that FA proteins are functionally involved in several complex cellular pathways including transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

  20. Artificial membranes with selective nanochannels for protein transport

    KAUST Repository

    Sutisna, Burhannudin

    2016-09-05

    A poly(styrene-b-tert-butoxystyrene-b-styrene) copolymer was synthesized by anionic polymerization and hydrolyzed to poly(styrene-b-4-hydroxystyrene-b-styrene). Lamellar morphology was confirmed in the bulk after annealing. Membranes were fabricated by self-assembly of the hydrolyzed copolymer in solution, followed by water induced phase separation. A high density of pores of 4 to 5 nm diameter led to a water permeance of 40 L m−2 h−1 bar−1 and molecular weight cut-off around 8 kg mol−1. The morphology was controlled by tuning the polymer concentration, evaporation time, and the addition of imidazole and pyridine to stabilize the terpolymer micelles in the casting solution via hydrogen bond complexes. Transmission electron microscopy of the membrane cross-sections confirmed the formation of channels with hydroxyl groups beneficial for hydrogen-bond forming sites. The morphology evolution was investigated by time-resolved grazing incidence small angle X-ray scattering experiments. The membrane channels reject polyethylene glycol with a molecular size of 10 kg mol−1, but are permeable to proteins, such as lysozyme (14.3 kg mol−1) and cytochrome c (12.4 kg mol−1), due to the right balance of hydrogen bond interactions along the channels, electrostatic attraction, as well as the right pore sizes. Our results demonstrate that artificial channels can be designed for protein transport via block copolymer self-assembly using classical methods of membrane preparation.

  1. Artificial membranes with selective nanochannels for protein transport

    KAUST Repository

    Sutisna, Burhannudin; Polymeropoulos, Georgios; Mygiakis, E.; Musteata, Valentina-Elena; Peinemann, Klaus-Viktor; Smilgies, D. M.; Hadjichristidis, Nikolaos; Nunes, Suzana Pereira

    2016-01-01

    A poly(styrene-b-tert-butoxystyrene-b-styrene) copolymer was synthesized by anionic polymerization and hydrolyzed to poly(styrene-b-4-hydroxystyrene-b-styrene). Lamellar morphology was confirmed in the bulk after annealing. Membranes were fabricated by self-assembly of the hydrolyzed copolymer in solution, followed by water induced phase separation. A high density of pores of 4 to 5 nm diameter led to a water permeance of 40 L m−2 h−1 bar−1 and molecular weight cut-off around 8 kg mol−1. The morphology was controlled by tuning the polymer concentration, evaporation time, and the addition of imidazole and pyridine to stabilize the terpolymer micelles in the casting solution via hydrogen bond complexes. Transmission electron microscopy of the membrane cross-sections confirmed the formation of channels with hydroxyl groups beneficial for hydrogen-bond forming sites. The morphology evolution was investigated by time-resolved grazing incidence small angle X-ray scattering experiments. The membrane channels reject polyethylene glycol with a molecular size of 10 kg mol−1, but are permeable to proteins, such as lysozyme (14.3 kg mol−1) and cytochrome c (12.4 kg mol−1), due to the right balance of hydrogen bond interactions along the channels, electrostatic attraction, as well as the right pore sizes. Our results demonstrate that artificial channels can be designed for protein transport via block copolymer self-assembly using classical methods of membrane preparation.

  2. Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases.

    Science.gov (United States)

    Brady, Scott T; Morfini, Gerardo A

    2017-09-01

    Neurons affected in a wide variety of unrelated adult-onset neurodegenerative diseases (AONDs) typically exhibit a "dying back" pattern of degeneration, which is characterized by early deficits in synaptic function and neuritic pathology long before neuronal cell death. Consistent with this observation, multiple unrelated AONDs including Alzheimer's disease, Parkinson's disease, Huntington's disease, and several motor neuron diseases feature early alterations in kinase-based signaling pathways associated with deficits in axonal transport (AT), a complex cellular process involving multiple intracellular trafficking events powered by microtubule-based motor proteins. These pathogenic events have important therapeutic implications, suggesting that a focus on preservation of neuronal connections may be more effective to treat AONDs than addressing neuronal cell death. While the molecular mechanisms underlying AT abnormalities in AONDs are still being analyzed, evidence has accumulated linking those to a well-established pathological hallmark of multiple AONDs: altered patterns of neuronal protein phosphorylation. Here, we present a short overview on the biochemical heterogeneity of major motor proteins for AT, their regulation by protein kinases, and evidence revealing cell type-specific AT specializations. When considered together, these findings may help explain how independent pathogenic pathways can affect AT differentially in the context of each AOND. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. In silico characterization of boron transporter (BOR1 protein sequences in Poaceae species

    Directory of Open Access Journals (Sweden)

    Ertuğrul Filiz

    2013-01-01

    Full Text Available Boron (B is essential for the plant growth and development, and its primary function is connected with formation of the cell wall. Moreover, boron toxicity is a shared problem in semiarid and arid regions. In this study, boron transporter protein (BOR1 sequences from some Poaceae species (Hordeum vulgare subsp. vulgare, Zea mays, Brachypodium distachyon, Oryza sativa subsp. japonica, Oryza sativa subsp. indica, Sorghum bicolor, Triticum aestivum were evaluated by bioinformatics tools. Physicochemical analyses revealed that most of BOR1 proteins were basic character and had generally aliphatic amino acids. Analysis of the domains showed that transmembrane domains were identified constantly and three motifs were detected with 50 amino acids length. Also, the motif SPNPWEPGSYDHWTVAKDMFNVPPAYIFGAFIPATMVAGLYYFDHSVASQ was found most frequently with 25 repeats. The phylogenetic tree showed divergence into two main clusters. B. distachyon species were clustered separately. Finally, this study contributes to the new BOR1 protein characterization in grasses and create scientific base for in silico analysis in future.

  4. Chemical shift homology in proteins

    International Nuclear Information System (INIS)

    Potts, Barbara C.M.; Chazin, Walter J.

    1998-01-01

    The degree of chemical shift similarity for homologous proteins has been determined from a chemical shift database of over 50 proteins representing a variety of families and folds, and spanning a wide range of sequence homologies. After sequence alignment, the similarity of the secondary chemical shifts of C α protons was examined as a function of amino acid sequence identity for 37 pairs of structurally homologous proteins. A correlation between sequence identity and secondary chemical shift rmsd was observed. Important insights are provided by examining the sequence identity of homologous proteins versus percentage of secondary chemical shifts that fall within 0.1 and 0.3 ppm thresholds. These results begin to establish practical guidelines for the extent of chemical shift similarity to expect among structurally homologous proteins

  5. Protein Restriction with Amino Acid-Balanced Diets Shrinks Circulating Pool Size of Amino Acid by Decreasing Expression of Specific Transporters in the Small Intestine.

    Directory of Open Access Journals (Sweden)

    Kai Qiu

    Full Text Available Dietary protein restriction is not only beneficial to health and longevity in humans, but also protects against air pollution and minimizes feeding cost in livestock production. However, its impact on amino acid (AA absorption and metabolism is not quite understood. Therefore, the study aimed to explore the effect of protein restriction on nitrogen balance, circulating AA pool size, and AA absorption using a pig model. In Exp.1, 72 gilts weighting 29.9 ± 1.5 kg were allocated to 1 of the 3 diets containing 14, 16, or 18% CP for a 28-d trial. Growth (n = 24, nitrogen balance (n = 6, and the expression of small intestinal AA and peptide transporters (n = 6 were evaluated. In Exp.2, 12 barrows weighting 22.7 ± 1.3 kg were surgically fitted with catheters in the portal and jejunal veins as well as the carotid artery and assigned to a diet containing 14 or 18% CP. A series of blood samples were collected before and after feeding for determining the pool size of circulating AA and AA absorption in the portal vein, respectively. Protein restriction did not sacrifice body weight gain and protein retention, since nitrogen digestibility was increased as dietary protein content reduced. However, the pool size of circulating AA except for lysine and threonine, and most AA flux through the portal vein were reduced in pigs fed the low protein diet. Meanwhile, the expression of peptide transporter 1 (PepT-1 was stimulated, but the expression of the neutral and cationic AA transporter systems was depressed. These results evidenced that protein restriction with essential AA-balanced diets, decreased AA absorption and reduced circulating AA pool size. Increased expression of small intestinal peptide transporter PepT-1 could not compensate for the depressed expression of jejunal AA transporters for AA absorption.

  6. BLAST-based structural annotation of protein residues using Protein Data Bank.

    Science.gov (United States)

    Singh, Harinder; Raghava, Gajendra P S

    2016-01-25

    In the era of next-generation sequencing where thousands of genomes have been already sequenced; size of protein databases is growing with exponential rate. Structural annotation of these proteins is one of the biggest challenges for the computational biologist. Although, it is easy to perform BLAST search against Protein Data Bank (PDB) but it is difficult for a biologist to annotate protein residues from BLAST search. A web-server StarPDB has been developed for structural annotation of a protein based on its similarity with known protein structures. It uses standard BLAST software for performing similarity search of a query protein against protein structures in PDB. This server integrates wide range modules for assigning different types of annotation that includes, Secondary-structure, Accessible surface area, Tight-turns, DNA-RNA and Ligand modules. Secondary structure module allows users to predict regular secondary structure states to each residue in a protein. Accessible surface area predict the exposed or buried residues in a protein. Tight-turns module is designed to predict tight turns like beta-turns in a protein. DNA-RNA module developed for predicting DNA and RNA interacting residues in a protein. Similarly, Ligand module of server allows one to predicted ligands, metal and nucleotides ligand interacting residues in a protein. In summary, this manuscript presents a web server for comprehensive annotation of a protein based on similarity search. It integrates number of visualization tools that facilitate users to understand structure and function of protein residues. This web server is available freely for scientific community from URL http://crdd.osdd.net/raghava/starpdb .

  7. A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.

    Science.gov (United States)

    Maerker, Tina; van Wijk, Erwin; Overlack, Nora; Kersten, Ferry F J; McGee, Joann; Goldmann, Tobias; Sehn, Elisabeth; Roepman, Ronald; Walsh, Edward J; Kremer, Hannie; Wolfrum, Uwe

    2008-01-01

    The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic analyses disclosed the colocalization of all network components in the apical inner segment collar and the ciliary apparatus of mammalian photoreceptor cells. In this complex, whirlin and SANS directly interact. Furthermore, SANS provides a linkage to the microtubule transport machinery, whereas whirlin may anchor USH2A isoform b and VLGR1b (very large G-protein coupled receptor 1b) via binding to their cytodomains at specific membrane domains. The long ectodomains of both transmembrane proteins extend into the gap between the adjacent membranes of the connecting cilium and the apical inner segment. Analyses of Vlgr1/del7TM mice revealed the ectodomain of VLGR1b as a component of fibrous links present in this gap. Comparative analyses of mouse and Xenopus photoreceptors demonstrated that this USH protein network is also part of the periciliary ridge complex in Xenopus. Since this structural specialization in amphibian photoreceptor cells defines a specialized membrane domain for docking and fusion of transport vesicles, we suggest a prominent role of the USH proteins in cargo shipment.

  8. Acid-base status determines the renal expression of Ca2+ and Mg2+ transport proteins.

    NARCIS (Netherlands)

    Nijenhuis, T.; Renkema, K.Y.; Hoenderop, J.G.J.; Bindels, R.J.M.

    2006-01-01

    Chronic metabolic acidosis results in renal Ca2+ and Mg2+ wasting, whereas chronic metabolic alkalosis is known to exert the reverse effects. It was hypothesized that these adaptations are mediated at least in part by the renal Ca2+ and Mg2+ transport proteins. The aim of this study, therefore, was

  9. Purinergic receptors stimulate Na+/Ca2+ exchange in pancreatic duct cells: possible role of proteins handling and transporting Ca2+

    DEFF Research Database (Denmark)

    Hansen, Mette R; Krabbe, Simon; Ankorina-Stark, Ieva

    2009-01-01

    ). Since NCX can also be connected with epithelial Ca(2+) transport, we also investigated expression of some Ca(2+)-handling/transporting proteins. Expression analysis revealed that pancreatic ducts of rat and human duct cell line CFPAC-1 (also PANC-1 and Capan-1) express the Na(+)/Ca(2+) exchanger (splice...

  10. Alkylsulfonates as probes of uncoupling protein transport mechanism. Ion pair transport demonstrates that direct H(+) translocation by UCP1 is not necessary for uncoupling

    Czech Academy of Sciences Publication Activity Database

    Jabůrek, M.; Vařecha, M.; Ježek, Petr; Garlid, K. D.

    2001-01-01

    Roč. 276, č. 34 (2001), s. 31897-31905 ISSN 0021-9258 R&D Projects: GA AV ČR IAA5011106 Grant - others:NIH(US) DK56273 Institutional research plan: CEZ:AV0Z5011922 Keywords : mitochondrial uncoupling proteins * alkylsulfonates * ion pair transport Subject RIV: CE - Biochemistry Impact factor: 7.258, year: 2001

  11. Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

    Directory of Open Access Journals (Sweden)

    Chao-Kuen Lai

    Full Text Available Nonstructural protein 5A (NS5A of hepatitis C virus (HCV serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

  12. Reliability of nine programs of topological predictions and their application to integral membrane channel and carrier proteins.

    Science.gov (United States)

    Reddy, Abhinay; Cho, Jaehoon; Ling, Sam; Reddy, Vamsee; Shlykov, Maksim; Saier, Milton H

    2014-01-01

    We evaluated topological predictions for nine different programs, HMMTOP, TMHMM, SVMTOP, DAS, SOSUI, TOPCONS, PHOBIUS, MEMSAT-SVM (hereinafter referred to as MEMSAT), and SPOCTOPUS. These programs were first evaluated using four large topologically well-defined families of secondary transporters, and the three best programs were further evaluated using topologically more diverse families of channels and carriers. In the initial studies, the order of accuracy was: SPOCTOPUS > MEMSAT > HMMTOP > TOPCONS > PHOBIUS > TMHMM > SVMTOP > DAS > SOSUI. Some families, such as the Sugar Porter Family (2.A.1.1) of the Major Facilitator Superfamily (MFS; TC #2.A.1) and the Amino Acid/Polyamine/Organocation (APC) Family (TC #2.A.3), were correctly predicted with high accuracy while others, such as the Mitochondrial Carrier (MC) (TC #2.A.29) and the K(+) transporter (Trk) families (TC #2.A.38), were predicted with much lower accuracy. For small, topologically homogeneous families, SPOCTOPUS and MEMSAT were generally most reliable, while with large, more diverse superfamilies, HMMTOP often proved to have the greatest prediction accuracy. We next developed a novel program, TM-STATS, that tabulates HMMTOP, SPOCTOPUS or MEMSAT-based topological predictions for any subdivision (class, subclass, superfamily, family, subfamily, or any combination of these) of the Transporter Classification Database (TCDB; www.tcdb.org) and examined the following subclasses: α-type channel proteins (TC subclasses 1.A and 1.E), secreted pore-forming toxins (TC subclass 1.C) and secondary carriers (subclass 2.A). Histograms were generated for each of these subclasses, and the results were analyzed according to subclass, family and protein. The results provide an update of topological predictions for integral membrane transport proteins as well as guides for the development of more reliable topological prediction programs, taking family-specific characteristics into account. © 2014 S. Karger AG, Basel.

  13. Artificial oxygen transport protein

    Science.gov (United States)

    Dutton, P. Leslie

    2014-09-30

    This invention provides heme-containing peptides capable of binding molecular oxygen at room temperature. These compounds may be useful in the absorption of molecular oxygen from molecular oxygen-containing atmospheres. Also included in the invention are methods for treating an oxygen transport deficiency in a mammal.

  14. Secondary gamma-ray data for shielding calculation

    International Nuclear Information System (INIS)

    Miyasaka, Sunichi

    1979-01-01

    In deep penetration transport calculations, the integral design parameters is determined mainly by secondary particles which are produced by interactions of the primary radiation with materials. The shield thickness and the biological dose rate at a given point of a bulk shield are determined from the contribution from secondary gamma rays. The heat generation and the radiation damage in the structural and shield materials depend strongly on the secondary gamma rays. In this paper, the status of the secondary gamma ray data and its further problems are described from the viewpoint of shield design. The secondary gamma-ray data in ENDF/B-IV and POPOP4 are also discussed based on the test calculations made for several shield assemblies. (author)

  15. Lignin, mitochondrial family and photorespiratory transporter classification as case studies in using co-expression, co-response and protein locations to aid in identifying transport functions

    Directory of Open Access Journals (Sweden)

    Takayuki eTohge

    2014-03-01

    Full Text Available Whole genome sequencing and the relative ease of transcript profiling have facilitated the collection and data warehousing of immense quantities of expression data. However, a substantial proportion of genes are not yet functionally annotated a problem which is particularly acute for transport proteins. In Arabidopsis, for example, only a minor fraction of the estimated 700 intracellular transporters have been identified at the molecular genetic level. Furthermore it is only within the last couple of years that critical genes such as those encoding the final transport step required for the long distance transport of sucrose and the first transporter of the core photorespiratory pathway have been identified. Here we will describe how transcriptional coordination between genes of known function and non-annotated genes allows the identification of putative transporters on the premise that such co-expressed genes tend to be functionally related. We will additionally extend this to include the expansion of this approach to include phenotypic information from other levels of cellular organization such as proteomic and metabolomic data and provide case studies wherein this approach has successfully been used to fill knowledge gaps in important metabolic pathways and physiological processes.

  16. Altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning

    NARCIS (Netherlands)

    J.A. Cabrera (Jesús); E.A. Ziemba (Elizabeth); L.H. Colbert (Lisa); L.B. Anderson (Lorraine); W.J. Sluiter (Wim); D.J.G.M. Duncker (Dirk); T.A. Butterick (Tammy); J. Sikora (Joseph); H.B. Ward (Herbert B.); R.F. Kelly (Rosemary); E.O. McFalls (Edward)

    2012-01-01

    textabstractAltered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated

  17. Transport of proteolipid protein to the plasma membrane does not depend on glycosphingolipid cotransport in oligodendrocyte cultures

    NARCIS (Netherlands)

    van der Haar, ME; Visser, HW; de Vries, H; Hoekstra, D

    1998-01-01

    The possibility that transport of proteolipid protein (PLP) from its site of synthesis to the plasma membrane is dependent on cotransport with (sulfo)galactocerebrosides was investigated in primary cultured oligodendrocytes and Chinese hamster ovary (CHO) cells expressing PLP. Sulfation was

  18. Interaction Of Calcium Phosphate Nanoparticles With Human Chorionic Gonadotropin Modifies Secondary And Tertiary Protein Structure

    Directory of Open Access Journals (Sweden)

    Al-Hakeim Hussein K

    2015-12-01

    Full Text Available Calcium phosphate nanoparticles (CaPNP have good biocompatibility and bioactivity inside human body. In this study, the interaction between CaPNP and human chorionic gonadotropin (hCG was analyzed to determine the changes in the protein structure in the presence of CaPNP and the quantity of protein adsorbed on the CaPNP surface. The results showed a significant adsorption of hCG on the CaPNP nanoparticle surface. The optimal fit was achieved using the Sips isotherm equation with a maximum adsorption capacity of 68.23 µg/mg. The thermodynamic parameters, including ∆H° and ∆G°, of the adsorption process are positive, whereas ∆S° is negative. The circular dichroism results of the adsorption of hCG on CaPNP showed the changes in its secondary structure; such changes include the decomposition of α-helix strand and the increase in β-pleated sheet and random coil percentages. Fluorescence study indicated minimal changes in the tertiary structure near the microenvironment of the aromatic amino acids such as tyrosine and phenyl alanine caused by the interaction forces between the CaPNP and hCG protein. The desorption process showed that the quantity of the hCG desorbed significantly increases as temperature increases, which indicates the weak forces between hCG and the surface.

  19. Transport Theory for Kinetic Emission of Secondary Electrons from Solids

    DEFF Research Database (Denmark)

    Schou, Jørgen

    1980-01-01

    a solid is derived. To find the former, existing computations for ion slowing down and experimental and theoretical ones for electron bombardment can be utilized. The energy and angular distribution of the secondary electrons and the secondary electron yield are both expressed as products of the deposited...... in the keV region is largely taken into account. The predicted energy and angular distribution agree with absolute spectra for incident electrons, whereas the agreement with absolute spectra for incident protons is less satisfactory. Extrapolation of the energy distribution down to the vacuum level gives...

  20. Contraction-stimulated glucose transport in muscle is controlled by AMPK and mechanical stress but not sarcoplasmatic reticulum Ca2+ release

    Directory of Open Access Journals (Sweden)

    Thomas E. Jensen

    2014-10-01

    Full Text Available Understanding how muscle contraction orchestrates insulin-independent muscle glucose transport may enable development of hyperglycemia-treating drugs. The prevailing concept implicates Ca2+ as a key feed forward regulator of glucose transport with secondary fine-tuning by metabolic feedback signals through proteins such as AMPK. Here, we demonstrate in incubated mouse muscle that Ca2+ release is neither sufficient nor strictly necessary to increase glucose transport. Rather, the glucose transport response is associated with metabolic feedback signals through AMPK, and mechanical stress-activated signals. Furthermore, artificial stimulation of AMPK combined with passive stretch of muscle is additive and sufficient to elicit the full contraction glucose transport response. These results suggest that ATP-turnover and mechanical stress feedback are sufficient to fully increase glucose transport during muscle contraction, and call for a major reconsideration of the established Ca2+ centric paradigm.

  1. Golgi localized barley MTP8 proteins facilitate Mn transport

    DEFF Research Database (Denmark)

    Pedas, Pai Rosager; Schiller, Michaela; Hegelund, Josefine Nymark

    2014-01-01

    Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2 , which encode membrane...... in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts...

  2. Elevated level of human RPA interacting protein α (hRIPα) in cervical tumor cells is involved in cell proliferation through regulating RPA transport.

    Science.gov (United States)

    Namkoong, Sim; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

    2012-10-19

    Replication protein A (RPA) is a eukaryotic single-stranded DNA binding protein that is essential for DNA replication, repair, and recombination, and human RPA interacting protein α (hRIPα) is the nuclear transporter of RPA. Here, we report the regulatory role of hRIPα protein in cell proliferation. Western blot analysis revealed that the level of hRIPα was frequently elevated in cervical tumors tissues and hRIPα knockdown by siRNA inhibited cellular proliferation through deregulation of the cell cycle. In addition, overexpression of hRIPα resulted in increased clonogenicity. These results indicate that hRIPα is involved in cell proliferation through regulation of RPA transport. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Low-Protein Diet Supplemented with Keto Acids Is Associated with Suppression of Small-Solute Peritoneal Transport Rate in Peritoneal Dialysis Patients

    Directory of Open Access Journals (Sweden)

    Na Jiang

    2011-01-01

    Full Text Available Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6–0.8 g/kg/d, keto-acid-supplemented low- (sLP: 0.6–0.8 g/kg/d with 0.12 g/kg/d of keto acids, or high- (HP: 1.0–1.2 g/kg/d protein diet and lasted for one year. In this study, the variations of peritoneal transport rate were assessed. Results. While baseline D/Pcr (dialysate-to-plasma concentration ratio for creatinine at 4 hour and D/D0glu (dialysate glucose at 4 hour to baseline dialysate glucose concentration ratio were similar, D/Pcr in group sLP was lower, and D/D0glu was higher than those in the other two groups (P<0.05 at 12th month. D/D0glu increased (P<0.05, and D/Pcr tended to decrease, (P=0.071 in group sLP. Conclusions. Low-protein diet with keto acids may benefit PD patients by maintaining peritoneum at a lower transport rate.

  4. Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

    Science.gov (United States)

    Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

    2018-06-01

    G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

  5. Low-protein diet supplemented with keto acids is associated with suppression of small-solute peritoneal transport rate in peritoneal dialysis patients.

    Science.gov (United States)

    Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Zhang, Weiming; Cao, Liou; Wang, Qin; Ni, Zhaohui; Yao, Qiang

    2011-01-01

    Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD) patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6-0.8 g/kg/d), keto-acid-supplemented low- (sLP: 0.6-0.8 g/kg/d with 0.12 g/kg/d of keto acids), or high- (HP: 1.0-1.2 g/kg/d) protein diet and lasted for one year. In this study, the variations of peritoneal transport rate were assessed. Results. While baseline D/P(cr) (dialysate-to-plasma concentration ratio for creatinine at 4 hour) and D/D0(glu) (dialysate glucose at 4 hour to baseline dialysate glucose concentration ratio) were similar, D/P(cr) in group sLP was lower, and D/D0(glu) was higher than those in the other two groups (P diet with keto acids may benefit PD patients by maintaining peritoneum at a lower transport rate.

  6. Effect of Secondary Doping Using Sorbitol on Structure and Transport Properties of PEDOT-PSS Thin Films

    Science.gov (United States)

    Khasim, Syed; Pasha, Apsar; Roy, Aashish S.; Parveen, Ameena; Badi, Nacer

    2017-07-01

    Poly(3,4-ethylene dioxythiophene):poly(styrenesulphonate) (PEDOT-PSS) in the recent past has emerged as one of the most fascinating conducting polymers for many device applications. The unique feature of PEDOT-PSS is its transparency in the entire visible spectrum with excellent thermal stability. The PEDOT-PSS as prepared as an aqueous dispersion has very low conductivity, and it hinders the performance of a device. In this work we report the conductivity enhancement of PEDOT-PSS thin films through secondary doping using a polar organic solvent such as sorbitol. The mechanism of conductivity enhancement was studied through various physical and chemical characterizations. The effect of sorbitol concentration on structure and transport properties of PEDOT-PSS thin films was investigated in detail. The structural and morphological modifications in PEDOT-PSS due to the addition of sorbitol was studied through Fourier transform spectroscopy, Ultra Violet-visible spectroscopy, theromogravimetric analysis, scanning electron microscopy and atomic force microscopy. The interactions resulting from conformational changes of PEDOT chains that changes from coiled to linear structure due to the sorbitol treatment significantly improves the conductivity of PEDOT-PSS films. The secondary doping of sorbitol reduces the energy barrier that facilitates the charge carrier hopping leading to enhanced conductivity. We have observed that the conductivity of PEDOT-PSS thin films was increased by two fold due to sorbitol treatment when compared to conductivity of pure PEDOT-PSS. We have carried out detailed analysis of dielectric parameters of sorbitol-treated PEDOT-PSS films and found that sorbitol treatment has a significant effect on various dielectric attributes of PEDOT-PSS films. Hence, secondary doping using sorbitol could be a useful way to effectively tailor the conductivity and dielectric properties of PEDOT-PSS thin films that can be used as flexible electrodes in

  7. mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli

    International Nuclear Information System (INIS)

    Zhang Weici; Xiao Weihua; Wei Haiming; Zhang Jian; Tian Zhigang

    2006-01-01

    Codon usage and thermodynamic optimization of the 5'-end of mRNA have been applied to improve the efficiency of human protein production in Escherichia coli. However, high level expression of human protein in E. coli is still a challenge that virtually depends upon each individual target genes. Using human interleukin 10 (huIL-10) and interferon α (huIFN-α) coding sequences, we systematically analyzed the influence of several major factors on expression of human protein in E. coli. The results from huIL-10 and reinforced by huIFN-α showed that exposing AUG initiator codon from base-paired structure within mRNA itself significantly improved the translation of target protein, which resulted in a 10-fold higher protein expression than the wild-type genes. It was also noted that translation process was not affected by the retained short-range stem-loop structure at Shine-Dalgarno (SD) sequences. On the other hand, codon-optimized constructs of huIL-10 showed unimproved levels of protein expression, on the contrary, led to a remarkable RNA degradation. Our study demonstrates that exposure of AUG initiator codon from long-range intra-strand secondary structure at 5'-end of mRNA may be used as a general strategy for human protein production in E. coli

  8. The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter

    OpenAIRE

    Nagai, Hiroki; Roy, Craig R.

    2001-01-01

    Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted Do...

  9. Transporter Classification Database (TCDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Transporter Classification Database details a comprehensive classification system for membrane transport proteins known as the Transporter Classification (TC)...

  10. Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.

    Directory of Open Access Journals (Sweden)

    Wenhan Zhu

    2011-03-01

    Full Text Available A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.

  11. Empirical correlation between protein backbone {sup 15}N and {sup 13}C secondary chemical shifts and its application to nitrogen chemical shift re-referencing

    Energy Technology Data Exchange (ETDEWEB)

    Wang Liya [Cold Spring Harbor Laboratory (United States); Markley, John L. [University of Wisconsin, Biochemistry Department (United States)], E-mail: markley@nmrfam.wisc.edu

    2009-06-15

    The linear analysis of chemical shifts (LACS) has provided a robust method for identifying and correcting {sup 13}C chemical shift referencing problems in data from protein NMR spectroscopy. Unlike other approaches, LACS does not require prior knowledge of the three-dimensional structure or inference of the secondary structure of the protein. It also does not require extensive assignment of the NMR data. We report here a way of extending the LACS approach to {sup 15}N NMR data from proteins, so as to enable the detection and correction of inconsistencies in chemical shift referencing for this nucleus. The approach is based on our finding that the secondary {sup 15}N chemical shift of the backbone nitrogen atom of residue i is strongly correlated with the secondary chemical shift difference (experimental minus random coil) between the alpha and beta carbons of residue i - 1. Thus once alpha and beta {sup 13}C chemical shifts are available (their difference is referencing error-free), the {sup 15}N referencing can be validated, and an appropriate offset correction can be derived. This approach can be implemented prior to a structure determination and can be used to analyze potential referencing problems in database data not associated with three-dimensional structure. Application of the LACS algorithm to the current BMRB protein chemical shift database, revealed that nearly 35% of the BMRB entries have {delta}{sup 15}N values mis-referenced by over 0.7 ppm and over 25% of them have {delta}{sup 1}H{sup N} values mis-referenced by over 0.12 ppm. One implication of the findings reported here is that a backbone {sup 15}N chemical shift provides a better indicator of the conformation of the preceding residue than of the residue itself.

  12. TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis.

    Science.gov (United States)

    Fukuchi, Junichi; Hiipakka, Richard A; Kokontis, John M; Nishimura, Kazuhiro; Igarashi, Kazuei; Liao, Shutsung

    2004-07-16

    Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

  13. Dynamic factors affecting gaseous ligand binding in an artificial oxygen transport protein.

    Science.gov (United States)

    Zhang, Lei; Andersen, Eskil M E; Khajo, Abdelahad; Magliozzo, Richard S; Koder, Ronald L

    2013-01-22

    We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7, this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities, and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime that may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when exposed to oxygen. Compared to that of HP7, the distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off rate. Electron paramagnetic resonance comparison of these ferric hemoproteins demonstrates that the mutation increases the level of disorder at the heme binding site. Nuclear magnetic resonance-detected deuterium exchange demonstrates that the mutation greatly increases the degree of penetration of water into the protein core. The inability of the mutant protein to bind oxygen may be due to an increased level of water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together, these data underline the importance of the control of protein dynamics in the design of functional artificial proteins.

  14. Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C' scalar couplings (3hbJNC')

    Energy Technology Data Exchange (ETDEWEB)

    Bonvin, Alexandre M.J.J.; Houben, Klaartje; Guenneugues, Marc; Kaptein, Robert; Boelens, Rolf [Utrecht University, Bijvoet Center for Biomolecular Research, NMR Spectroscopy (Netherlands)

    2001-11-15

    The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small {alpha}/{beta} protein chymotrypsin inhibitor 2. Dihedral angle restraints for the {phi} and {psi} angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond {sup 3hb}J{sub NC'} coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.

  15. The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

    KAUST Repository

    Benedet, Mattia; Falchi, Federica A.; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra; Deho, Gianni

    2016-01-01

    The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in gamma-Proteobacteria. LptBFG constitute the IMABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable Delta lptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptF(SupC)). In complementation tests, lptF(SupC) mutants suppress lethality of both Delta lptC and lptC conditional expressionmutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

  16. The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

    KAUST Repository

    Benedet, Mattia

    2016-08-16

    The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in gamma-Proteobacteria. LptBFG constitute the IMABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable Delta lptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptF(SupC)). In complementation tests, lptF(SupC) mutants suppress lethality of both Delta lptC and lptC conditional expressionmutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

  17. Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information

    International Nuclear Information System (INIS)

    Fritzsching, K. J.; Yang, Y.; Schmidt-Rohr, K.; Hong Mei

    2013-01-01

    We introduce a Python-based program that utilizes the large database of 13 C and 15 N chemical shifts in the Biological Magnetic Resonance Bank to rapidly predict the amino acid type and secondary structure from correlated chemical shifts. The program, called PACSYlite Unified Query (PLUQ), is designed to help assign peaks obtained from 2D 13 C– 13 C, 15 N– 13 C, or 3D 15 N– 13 C– 13 C magic-angle-spinning correlation spectra. We show secondary-structure specific 2D 13 C– 13 C correlation maps of all twenty amino acids, constructed from a chemical shift database of 262,209 residues. The maps reveal interesting conformation-dependent chemical shift distributions and facilitate searching of correlation peaks during amino-acid type assignment. Based on these correlations, PLUQ outputs the most likely amino acid types and the associated secondary structures from inputs of experimental chemical shifts. We test the assignment accuracy using four high-quality protein structures. Based on only the Cα and Cβ chemical shifts, the highest-ranked PLUQ assignments were 40–60 % correct in both the amino-acid type and the secondary structure. For three input chemical shifts (CO–Cα–Cβ or N–Cα–Cβ), the first-ranked assignments were correct for 60 % of the residues, while within the top three predictions, the correct assignments were found for 80 % of the residues. PLUQ and the chemical shift maps are expected to be useful at the first stage of sequential assignment, for combination with automated sequential assignment programs, and for highly disordered proteins for which secondary structure analysis is the main goal of structure determination.

  18. Regulation of auxin transport during gravitropism

    Science.gov (United States)

    Rashotte, A.; Brady, S.; Kirpalani, N.; Buer, C.; Muday, G.

    Plants respond to changes in the gravity vector by differential growth across the gravity-stimulated organ. The plant hormone auxin, which is normally basipetally transported, changes in direction and auxin redistribution has been suggested to drive this differential growth or gravitropism. The mechanisms by which auxin transport directionality changes in response to a change in gravity vector are largely unknown. Using the model plant, Arabidopsis thaliana, we have been exploring several regulatory mechanisms that may control auxin transport. Mutations that alter protein phosphorylation suggest that auxin transport in arabidopsis roots may be controlled via phosphorylation and this signal may facilitate gravitropic bending. The protein kinase mutant pinoid (pid9) has reduced auxin transport; whereas the protein phosphatase mutant, rcn1, has elevated transport, suggesting reciprocal regulation of auxin transport by reversible protein phosphorylation. In both of these mutants, the auxin transport defects are accompanied by gravitropic defects, linking phosphorylation signaling to gravity-induced changes in auxin transport. Additionally, auxin transport may be regulated during gravity response by changes in an endogenous auxin efflux inhibitor. Flavonoids, such as quercetin and kaempferol, have been implicated in regulation of auxin transport in vivo and in vitro. Mutants that make no flavonoids have reduced root gravitropic bending. Furthermore, changes in auxin-induced gene expression and flavonoid accumulation patterns have been observed during gravity stimulation. Current studies are examining whether there are spatial and temporal changes in flavonoid accumulation that precede gravitropic bending and whether the absence of these changes are the cause of the altered gravity response in plants with mutations that block flavonoid synthesis. These results support the idea that auxin transport may be regulated during gravity response by several mechanisms including

  19. Classification of methods and equipment recovery secondary waters

    Directory of Open Access Journals (Sweden)

    G. V. Kalashnikov

    2017-01-01

    Full Text Available The issues of purification of secondary waters of industrial production have an important place and are relevant in the environmental activities of all food and chemical industries. For cleaning the transporter-washing water of beet-sugar production the key role is played by the equipment of treatment plants. A wide variety of wastewater treatment equipment is classified according to various methods. Typical structures used are sedimentation tanks, hydrocyclones, separators, centrifuges. In turn, they have a different degree of purification, productivity through the incoming suspension and purified secondary water. This is equipment is divided into designs, depending on the range of particles to be removed. A general classification of methods for cleaning the transporter-washing water, as well as the corresponding equipment, is made. Based on the analysis of processes and instrumentation, the main methods of wastewater treatment are identified: mechanical, physicochemical, combined, biological and disinfection. To increase the degree of purification and reduce technical and economic costs, a combined method is widely used. The main task of the site for cleaning the transporter-washing waters of sugar beet production is to provide the enterprise with water in the required quantity and quality, with economical use of water resources, taking into account the absence of pollution of surface and groundwater by industrial wastewater. In the sugar industry is currently new types of washing equipment of foreign production are widely used, which require high quality and a large amount of purified transporter-washing water for normal operation. The proposed classification makes it possible to carry out a comparative technical and economic analysis when choosing the methods and equipment for recuperation of secondary waters. The main equipment secondary water recovery used at the beet-sugar plant is considered. The most common beet processing plant is a

  20. Characterizing the Secondary Protein Structure of Black Widow Dragline Silk Using Solid-State NMR & X-ray Diffraction

    Science.gov (United States)

    Jenkins, Janelle E.; Sampath, Sujatha; Butler, Emily; Kim, Jihyun; Henning, Robert W.; Holland, Gregory P.; Yarger, Jeffery L.

    2013-01-01

    This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked β-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) 13C-13C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and hence to the amino acids that make up the motifs. Specifically, alanine is incorporated in β-sheet (poly(Alan) and poly(Gly-Ala)), 31-helix (poly(Gly-Gly-Xaa), and α-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in β-sheet (poly(Gly-Ala)) and 31-helical (poly(Gly-Gly-Xaa)) regions, while serine is present in β-sheet (poly(Gly-Ala-Ser)), 31-helix (poly(Gly-Gly-Ser)), and β-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk. PMID:24024617

  1. Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties ▿

    Science.gov (United States)

    Sánchez-Navarro, Jesús; Fajardo, Thor; Zicca, Stefania; Pallás, Vicente; Stavolone, Livia

    2010-01-01

    Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway. PMID:20130061

  2. GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary.

    Directory of Open Access Journals (Sweden)

    Chen Jiang

    2017-01-01

    Full Text Available Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate (GGPP, a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase (Ggpps levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function.

  3. An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

    Science.gov (United States)

    Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

    2016-01-01

    A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

  4. The riboflavin transporter RibU in Lactococcus lactis : Molecular characterization of gene expression and the transport mechanism

    NARCIS (Netherlands)

    Burgess, CM; Slotboom, DJ; Geertsma, ER; Duurkens, Hinderika; Poolman, B; van Sinderen, D

    This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport

  5. Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide

    Directory of Open Access Journals (Sweden)

    Hajime Yamazaki

    2017-06-01

    Full Text Available Previously, we have shown that serine-16 phosphorylation in native full-length porcine amelogenin (P173 and the Leucine-Rich Amelogenin Peptide (LRAP(+P, an alternative amelogenin splice product, affects protein assembly and mineralization in vitro. Notably, P173 and LRAP(+P stabilize amorphous calcium phosphate (ACP and inhibit hydroxyapatite (HA formation, while non-phosphorylated counterparts (rP172, LRAP(−P guide the growth of ordered bundles of HA crystals. Based on these findings, we hypothesize that the phosphorylation of full-length amelogenin and LRAP induces conformational changes that critically affect its capacity to interact with forming calcium phosphate mineral phases. To test this hypothesis, we have utilized Fourier transform infrared spectroscopy (FTIR to determine the secondary structure of LRAP(−P and LRAP(+P in the absence/presence of calcium and selected mineral phases relevant to amelogenesis; i.e., hydroxyapatite (HA: an enamel crystal prototype and (ACP: an enamel crystal precursor phase. Aqueous solutions of LRAP(−P or LRAP(+P were prepared with or without 7.5 mM of CaCl2 at pH 7.4. FTIR spectra of each solution were obtained using attenuated total reflectance, and amide-I peaks were analyzed to provide secondary structure information. Secondary structures of LRAP(+P and LRAP(−P were similarly assessed following incubation with suspensions of HA and pyrophosphate-stabilized ACP. Amide I spectra of LRAP(−P and LRAP(+P were found to be distinct from each other in all cases. Spectra analyses showed that LRAP(−P is comprised mostly of random coil and β-sheet, while LRAP(+P exhibits more β-sheet and α-helix with little random coil. With added Ca, the random coil content increased in LRAP(−P, while LRAP(+P exhibited a decrease in α-helix components. Incubation of LRAP(−P with HA or ACP resulted in comparable increases in β-sheet structure. Notably, however, LRAP(+P secondary structure was more affected by

  6. Molecular properties of mammalian proteins that interact with cGMP: protein kinases, cation channels, phosphodiesterases, and multi-drug anion transporters.

    Science.gov (United States)

    Francis, Sharron H; Blount, Mitsi A; Zoraghi, Roya; Corbin, Jackie D

    2005-09-01

    Cyclic GMP is a critical second messenger signaling molecule in many mammalian cell types. It is synthesized by a family of guanylyl cyclases that is activated in response to stimuli from hormones such as natriuretic peptides, members of the guanylin family, and chemical stimuli including nitric oxide and carbon monoxide. The resulting elevation of cGMP modulates myriad physiological processes. Three major groups of cellular proteins bind cGMP specifically at allosteric sites; interaction of cGMP with these sites modulates the activities and functions of other domains within these protein groups to bring about physiological effects. These proteins include the cyclic nucleotide (cN)-dependent protein kinases, cN-gated cation channels, and cGMP-binding phosphodiesterases (PDE). Cyclic GMP also interacts with the catalytic sites of many cN PDEs and with some members of the multi-drug anion transporter family (MRPs) which can extrude nucleotides from cells. The allosteric cN-binding sites in the kinases and the cN-gated channels are evolutionarily and biochemically related, whereas the allosteric cGMP-binding sites in PDEs (also known as GAF domains), the catalytic sites of PDEs , and the ligand-binding sites in the MRPs are evolutionarily and biochemically distinct from each other and from those in the kinase and channel families. The sites that interact with cGMP within each of these groups of proteins have unique properties that provide for cGMP binding. Within a given cell, cGMP can potentially interact with members of all these groups of proteins if they are present. The relative abundance and affinities of these various cGMP-binding sites in conjunction with their subcellular compartmentation, proximity to cyclases and PDEs, and post-translational modification contribute importantly in determining the impact of these respective proteins to cGMP signaling within a particular cell.

  7. EXTRACELLULAR PROTEINS PRODUCED BY DIFFERENT SPECIES OF THE FUNGUS TRICHODERMA ON SECONDARY PAPER MILL SLUDGE SUBSTRATE

    Directory of Open Access Journals (Sweden)

    Ida Vaskova,

    2012-01-01

    Full Text Available Kraft pulping is the most commonly used pulping process in the pulp and paper industry. In this process wood chips are chemically delignified using sodium sulfide and sodium hydroxide. Delignification is usually followed by mechanical fiberization and a bleaching process of the resulting wood pulp. In addition to lignin-free wood pulp, this process also produces waste that contains residues of used chemicals, lignin, cellulose, hemicelluloses, and small amounts of other wood components. Because of the worldwide large-scale production of paper, the sludge from paper mills contributes significantly to environmental pollution. Although there have been great efforts being made to utilize this lignin-rich material, sludge is mostly disposed in landfills or incinerated in a boiler. This research project used secondary sludge as a substrate for 7 wood-decay fungi taxonomically belonging to the genus Trichoderma. The examined fungi expressed the capability of consuming sludge components as a carbon source to produce extracellular proteins. The proteins were separated by gel electrophoresis. Before and after fungi cultivation, the sludge was analyzed by Fourier transform infrared spectroscopy (FTIR.

  8. Characterization of dextran-grafted hydrophobic charge-induction resins: Structural properties, protein adsorption and transport.

    Science.gov (United States)

    Liu, Tao; Angelo, James M; Lin, Dong-Qiang; Lenhoff, Abraham M; Yao, Shan-Jing

    2017-09-29

    The structural and functional properties of a series of dextran-grafted and non-grafted hydrophobic charge-induction chromatographic (HCIC) agarose resins were characterized by macroscopic and microscopic techniques. The effects of dextran grafting and mobile phase conditions on the pore dimensions of the resins were investigated with inverse size exclusion chromatography (ISEC). A significantly lower pore radius (17.6nm) was found for dextran-grafted than non-grafted resins (29.5nm), but increased salt concentration would narrow the gap between the respective pore radii. Two proteins, human immunoglobulin G (hIgG) and bovine serum albumin (BSA), were used to examine the effect of protein characteristics. The results of adsorption isotherms showed that the dextran-grafted resin with high ligand density had substantially higher adsorption capacity and enhanced the salt-tolerance property for hIgG, but displayed a significantly smaller benefit for BSA adsorption. Confocal laser scanning microscopy (CLSM) showed that hIgG presented more diffuse and slower moving adsorption front compared to BSA during uptake into the resins because of the selective binding of multiple species from polyclonal IgG; polymer-grafting with high ligand density could enhance the rate of hIgG transport in the dextran-grafted resins without salt addition, but not for the case with high salt and BSA. The results indicate that microscopic analysis using ISEC and CLSM is useful to improve the mechanistic understanding of resin structure and of critical functional parameters involving protein adsorption and transport, which would guide the rational design of new resins and processes. Copyright © 2017. Published by Elsevier B.V.

  9. The Down regulated in Adenoma (dra) gene encodes an intestine-specific membrane sulfate transport protein.

    Science.gov (United States)

    Silberg, D G; Wang, W; Moseley, R H; Traber, P G

    1995-05-19

    A gene has been described, Down Regulated in Adenoma (dra), which is expressed in normal colon but is absent in the majority of colon adenomas and adenocarcinomas. However, the function of this protein is unknown. Because of sequence similarity to a recently cloned membrane sulfate transporter in rat liver, the transport function of Dra was examined. We established that dra encodes for a Na(+)-independent transporter for both sulfate and oxalate using microinjected Xenopus oocytes as an assay system. Sulfate transport was sensitive to the anion exchange inhibitor DIDS (4,4'-diisothiocyano-2,2' disulfonic acid stilbene). Using an RNase protection assay, we found that dra mRNA expression is limited to the small intestine and colon in mouse, therefore identifying Dra as an intestine-specific sulfate transporter. dra also had a unique pattern of expression during intestinal development. Northern blot analysis revealed a low level of expression in colon at birth with a marked increase in the first 2 postnatal weeks. In contrast, there was a lower, constant level of expression in small intestine in the postnatal period. Caco-2 cells, a colon carcinoma cell line that differentiates over time in culture, demonstrated a marked induction of dra mRNA as cells progressed from the preconfluent (undifferentiated) to the postconfluent (differentiated) state. These results show that Dra is an intestine-specific Na(+)-independent sulfate transporter that has differential expression during colonic development. This functional characterization provides the foundation for investigation of the role of Dra in intestinal sulfate transport and in the malignant phenotype.

  10. Effects of inhibitors of protein synthesis and intracellular transport on the gamma-aminobutyric acid agonist-induced functional differentiation of cultured cerebellar granule cells

    DEFF Research Database (Denmark)

    Belhage, B; Hansen, Gert Helge; Meier, E

    1990-01-01

    The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological...... an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low-affinity GABA receptors. In contrast, the inhibitors of intracellular transport...

  11. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  12. High-pressure EPR spectroscopy studies of the E. coli lipopolysaccharide transport proteins LptA and LptC.

    Science.gov (United States)

    Schultz, Kathryn M; Klug, Candice S

    2017-12-01

    The use of pressure is an advantageous approach to the study of protein structure and dynamics because it can shift the equilibrium populations of protein conformations toward higher energy states that are not of sufficient population to be observable at atmospheric pressure. Recently, the Hubbell group at the University of California, Los Angeles, reintroduced the application of high pressure to the study of proteins by electron paramagnetic resonance (EPR) spectroscopy. This methodology is possible using X-band EPR spectroscopy due to advances in pressure intensifiers, sample cells, and resonators. In addition to the commercial availability of the pressure generation and sample cells by Pressure Biosciences Inc., a five-loop-four-gap resonator required for the initial high pressure EPR spectroscopy experiments by the Hubbell group, and those reported here, was designed by James S. Hyde and built and modified at the National Biomedical EPR Center. With these technological advances, we determined the effect of pressure on the essential periplasmic lipopolysaccharide (LPS) transport protein from Escherichia coli , LptA, and one of its binding partners, LptC. LptA unfolds from the N-terminus to the C-terminus, binding of LPS does not appreciably stabilize the protein under pressure, and monomeric LptA unfolds somewhat more readily than oligomeric LptA upon pressurization to 2 kbar. LptC exhibits a fold and relative lack of stability upon LPS binding similar to LptA, yet adopts an altered, likely monomeric, folded conformation under pressure with only its C-terminus unraveling. The pressure-induced changes likely correlate with functional changes associated with binding and transport of LPS.

  13. Properties of the mitochondrial carrier of adenine-nucleotide after purification. Study of the transport protein under isolated form and reincorporated form in phospho-lipidic vesicles

    International Nuclear Information System (INIS)

    Brandolin, Gerard

    1983-01-01

    The first part of this research thesis addresses the reconstitution of the ADP/ATP transport by incorporation of the specific carrier, isolated in presence of detergent, in phospholipids vesicles. Fundamental properties of the reconstituted transport are identical to that of transport in mitochondria, notably as far as the exchange stoichiometry, the turn over and the transport Km are concerned, as well as the asymmetric orientation of the carrier in the membrane. The second part of this research addresses the study of interactions of specific ligands with the ADP/ATP transport protein in presence of detergent. The study of the variations of the intrinsic fluorescence of the isolated ADP/ATP carrier highlights conformational changes exclusively induced by the presence of transportable nucleotides which are modulated in a different manner by carboxy-atractyloside or bongkrekic acid. Moreover, by using the isolated protein, a detailed analysis of binding parameters of fluorescent analogues of ATP is reported [fr

  14. A universal DNA-based protein detection system.

    Science.gov (United States)

    Tran, Thua N N; Cui, Jinhui; Hartman, Mark R; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C; Lis, John T; Cui, Haixin; Luo, Dan

    2013-09-25

    Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

  15. The effects of secondary mineral precipitates on {sup 90}Sr mobility at the Hanford Site, USA

    Energy Technology Data Exchange (ETDEWEB)

    Um, Wooyong [Pacific Northwest National Laboratory, 902 Battelle Blvd., MSIN P7-54, Richland, WA 99354 (United States); Division of Advanced Nuclear Engineering, Pohang University of Science and Technology - POSTECH (Korea, Republic of); Wang, Guohui; Serne, R. Jeffrey [Pacific Northwest National Laboratory, 902 Battelle Blvd., MSIN P7-54, Richland, WA 99354 (United States)

    2013-07-01

    The effects of secondary precipitates on {sup 90}Sr transport at the Hanford Site were investigated using quartz column experiments with simulated caustic tank waste leachates (STWL). Significantly enhanced retardation of Sr transport was observed in the column contacted with STWL due to Sr sorption and co-precipitation with neo-formed nitrate cancrinite. However, the column results also suggest that neo-formed secondary precipitates could behave like native mobile colloids that can enhance Sr transport. Initially immobilized Sr within secondary precipitates could re-mobilize given a change in the pore water background conditions. The mobility of the neo-formed Sr-bearing precipitates increased with increased solution flow rate. In the field, pore water contents and flow rates can be changed by snow-melt (or storm water) events or artificial infiltration. The increased pore water flow rate caused by these events could affect the mobility of {sup 90}Sr-containing secondary precipitates, which can be a potential source for facilitated Sr transport in Hanford Site subsurface environments. (authors)

  16. The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins.

    Science.gov (United States)

    López-Carrasco, Amparo; Flores, Ricardo

    2017-07-01

    Avocado sunblotch viroid (ASBVd), the type member of the family Avsunviroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246-250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (-) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2'-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (-) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber viroid, the type member of nuclear viroids (family Pospiviroidae), indicating that two different viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected avocado leaves.

  17. Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information

    Energy Technology Data Exchange (ETDEWEB)

    Fritzsching, K. J.; Yang, Y.; Schmidt-Rohr, K.; Hong Mei, E-mail: mhong@iastate.edu [Iowa State University, Department of Chemistry (United States)

    2013-06-15

    We introduce a Python-based program that utilizes the large database of {sup 13}C and {sup 15}N chemical shifts in the Biological Magnetic Resonance Bank to rapidly predict the amino acid type and secondary structure from correlated chemical shifts. The program, called PACSYlite Unified Query (PLUQ), is designed to help assign peaks obtained from 2D {sup 13}C-{sup 13}C, {sup 15}N-{sup 13}C, or 3D {sup 15}N-{sup 13}C-{sup 13}C magic-angle-spinning correlation spectra. We show secondary-structure specific 2D {sup 13}C-{sup 13}C correlation maps of all twenty amino acids, constructed from a chemical shift database of 262,209 residues. The maps reveal interesting conformation-dependent chemical shift distributions and facilitate searching of correlation peaks during amino-acid type assignment. Based on these correlations, PLUQ outputs the most likely amino acid types and the associated secondary structures from inputs of experimental chemical shifts. We test the assignment accuracy using four high-quality protein structures. Based on only the C{alpha} and C{beta} chemical shifts, the highest-ranked PLUQ assignments were 40-60 % correct in both the amino-acid type and the secondary structure. For three input chemical shifts (CO-C{alpha}-C{beta} or N-C{alpha}-C{beta}), the first-ranked assignments were correct for 60 % of the residues, while within the top three predictions, the correct assignments were found for 80 % of the residues. PLUQ and the chemical shift maps are expected to be useful at the first stage of sequential assignment, for combination with automated sequential assignment programs, and for highly disordered proteins for which secondary structure analysis is the main goal of structure determination.

  18. Phosphorylation of Ribosomal Protein S6 Mediates Mammalian Target of Rapamycin Complex 1-Induced Parathyroid Cell Proliferation in Secondary Hyperparathyroidism.

    Science.gov (United States)

    Volovelsky, Oded; Cohen, Gili; Kenig, Ariel; Wasserman, Gilad; Dreazen, Avigail; Meyuhas, Oded; Silver, Justin; Naveh-Many, Tally

    2016-04-01

    Secondary hyperparathyroidism is characterized by increased serum parathyroid hormone (PTH) level and parathyroid cell proliferation. However, the molecular pathways mediating the increased parathyroid cell proliferation remain undefined. Here, we found that the mTOR pathway was activated in the parathyroid of rats with secondary hyperparathyroidism induced by either chronic hypocalcemia or uremia, which was measured by increased phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTOR pathway. This activation correlated with increased parathyroid cell proliferation. Inhibition of mTOR complex 1 by rapamycin decreased or prevented parathyroid cell proliferation in secondary hyperparathyroidism rats and in vitro in uremic rat parathyroid glands in organ culture. Knockin rpS6(p-/-) mice, in which rpS6 cannot be phosphorylated because of substitution of all five phosphorylatable serines with alanines, had impaired PTH secretion after experimental uremia- or folic acid-induced AKI. Uremic rpS6(p-/-) mice had no increase in parathyroid cell proliferation compared with a marked increase in uremic wild-type mice. These results underscore the importance of mTOR activation and rpS6 phosphorylation for the pathogenesis of secondary hyperparathyroidism and indicate that mTORC1 is a significant regulator of parathyroid cell proliferation through rpS6. Copyright © 2016 by the American Society of Nephrology.

  19. Transporter-mediated biofuel secretion.

    Science.gov (United States)

    Doshi, Rupak; Nguyen, Tuan; Chang, Geoffrey

    2013-05-07

    Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux lipids and drugs, can be used to mediate the secretion of intracellularly synthesized model isoprenoid biofuel compounds to the extracellular milieu. Transporter-mediated biofuel secretion sustainably maintained an approximate three- to fivefold boost in biofuel production in our Escherichia coli test system. Because the transporters used in this study belong to the ubiquitous ATP-binding cassette protein family, we propose their use as "plug-and-play" biofuel-secreting systems in a variety of bacteria, cyanobacteria, diatoms, yeast, and algae used for biofuel production. This investigation showcases the potential of expressing desired membrane transport proteins in cell factories to achieve the export or import of substances of economic, environmental, or therapeutic importance.

  20. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

    Science.gov (United States)

    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  1. Loss of C. elegans BBS-7 and BBS-8 protein function results in cilia defects and compromised intraflagellar transport

    OpenAIRE

    Blacque, Oliver E.; Reardon, Michael J.; Li, Chunmei; McCarthy, Jonathan; Mahjoub, Moe R.; Ansley, Stephen J.; Badano, Jose L.; Mah, Allan K.; Beales, Philip L.; Davidson, William S.; Johnsen, Robert C.; Audeh, Mark; Plasterk, Ronald H.A.; Baillie, David L.; Katsanis, Nicholas

    2004-01-01

    Bardet-Biedl syndrome (BBS) is a genetically heterogeneous developmental disorder whose molecular basis is largely unknown. Here, we show that mutations in the Caenorhabditis elegans bbs-7 and bbs-8 genes cause structural and functional defects in cilia. C. elegans BBS proteins localize predominantly at the base of cilia, and like proteins involved in intraflagellar transport (IFT), a process necessary for cilia biogenesis and maintenance, move bidirectionally along the ciliary axoneme. Impor...

  2. Cyanobacteria as Cell Factories to Produce Plant Secondary Metabolites

    OpenAIRE

    Xue, Yong; He, Qingfang

    2015-01-01

    Cyanobacteria represent a promising platform for the production of plant secondary metabolites. Their capacity to express plant P450 proteins, which have essential functions in the biosynthesis of many plant secondary metabolites, makes cyanobacteria ideal for this purpose, and their photosynthetic capability allows cyanobacteria to grow with simple nutrient inputs. This review summarizes the advantages of using cyanobacteria to transgenically produce plant secondary metabolites. Some techniq...

  3. Parental influenza virion nucleocapsids are efficiently transported into the nuclei of murine cells expressing the nuclear interferon-induced Mx protein.

    Science.gov (United States)

    Broni, B; Julkunen, I; Condra, J H; Davies, M E; Berry, M J; Krug, R M

    1990-12-01

    The interferon-induced murine Mx1 protein, which is localized in the nucleus, most likely specifically blocks influenza virus replication by inhibiting nuclear viral mRNA synthesis, including the mRNA synthesis catalyzed by inoculum (parental) virion nucleocapsids (R. M. Krug, M. Shaw, B. Broni, G. Shapiro, and O. Haller, J. Virol. 56:201-206, 1985). We tested two possible mechanisms for this inhibition. First, we determined whether the transport of parental nucleocapsids into the nucleus was inhibited in murine cells expressing the nuclear Mx1 protein. To detect the Mx1 protein, we prepared rabbit antibodies against the Mx1 protein with a CheY-Mx fusion protein expressed in bacteria. The fate of parental nucleocapsids was monitored by immunofluorescence with an appropriate dilution of monoclonal antibody to the nucleocapsid protein. The protein synthesis inhibitor anisomycin was added to the cells 30 min prior to infection, so that the only nucleocapsids protein molecules in the cells were those associated with nucleocapsids of the parental virus. These nucleocapsids were efficiently transported into the nuclei of murine cells expressing the Mx1 protein, indicating that this protein most likely acts after the parental nucleocapsids enter the nucleus. The second possibility was that the murine Mx1 protein might act in the nucleus to inhibit viral mRNA synthesis indirectly via new cap-binding activities that sequestered cellular capped RNAs away from the viral RNA transcriptase. We show that the same array of nuclear cap-binding proteins was present in Mx-positive and Mx-negative cells treated with interferon. Interestingly, a large amount of a 43-kDa cap-binding activity appeared after interferon treatment of both Mx-positive and Mx-negative cells. Hence, the appearance of new cap-binding activities was unlikely to account for the Mx-specific inhibition of viral mRNA synthesis. These results are most consistent with the possibility that the Mx1 protein acts

  4. Quaternary structure of the lactose transport protein of Streptococcus thermophilus in the detergent-solubilized and membrane-reconstituted state

    NARCIS (Netherlands)

    Friesen, R.H.E.; Poolman, B.; Knol, J.

    2000-01-01

    The quaternary structure of LacS, the lactose transporter of Streptococcus thermophilus, has been determined for the detergent-solubilized and the membrane-reconstituted state of the protein. The quaternary structure of the n-dodecyl-β-D-maltoside-solubilized state was studied using a combination of

  5. Medicago truncatula transporter database: a comprehensive database resource for M. truncatula transporters

    Directory of Open Access Journals (Sweden)

    Miao Zhenyan

    2012-02-01

    Full Text Available Abstract Background Medicago truncatula has been chosen as a model species for genomic studies. It is closely related to an important legume, alfalfa. Transporters are a large group of membrane-spanning proteins. They deliver essential nutrients, eject waste products, and assist the cell in sensing environmental conditions by forming a complex system of pumps and channels. Although studies have effectively characterized individual M. truncatula transporters in several databases, until now there has been no available systematic database that includes all transporters in M. truncatula. Description The M. truncatula transporter database (MTDB contains comprehensive information on the transporters in M. truncatula. Based on the TransportTP method, we have presented a novel prediction pipeline. A total of 3,665 putative transporters have been annotated based on International Medicago Genome Annotated Group (IMGAG V3.5 V3 and the M. truncatula Gene Index (MTGI V10.0 releases and assigned to 162 families according to the transporter classification system. These families were further classified into seven types according to their transport mode and energy coupling mechanism. Extensive annotations referring to each protein were generated, including basic protein function, expressed sequence tag (EST mapping, genome locus, three-dimensional template prediction, transmembrane segment, and domain annotation. A chromosome distribution map and text-based Basic Local Alignment Search Tools were also created. In addition, we have provided a way to explore the expression of putative M. truncatula transporter genes under stress treatments. Conclusions In summary, the MTDB enables the exploration and comparative analysis of putative transporters in M. truncatula. A user-friendly web interface and regular updates make MTDB valuable to researchers in related fields. The MTDB is freely available now to all users at http://bioinformatics.cau.edu.cn/MtTransporter/.

  6. Neurotransmitter transporters

    DEFF Research Database (Denmark)

    Gether, Ulrik; Andersen, Peter H; Larsson, Orla M

    2006-01-01

    The concentration of neurotransmitters in the extracellular space is tightly controlled by distinct classes of membrane transport proteins. This review focuses on the molecular function of two major classes of neurotransmitter transporter that are present in the cell membrane of neurons and....... Recent research has provided substantial insight into the structure and function of these transporters. In particular, the recent crystallizations of bacterial homologs are of the utmost importance, enabling the first reliable structural models of the mammalian neurotransmitter transporters...

  7. Inhibitors of plant hormone transport

    Czech Academy of Sciences Publication Activity Database

    Klíma, Petr; Laňková, Martina; Zažímalová, Eva

    2016-01-01

    Roč. 253, č. 6 (2016), s. 1391-1404 ISSN 0033-183X R&D Projects: GA MŠk(CZ) LD15088 Institutional support: RVO:61389030 Keywords : polar auxin transport * acid-binding protein * gnom arf-gef * equilibrative nucleoside transporter * efflux carrier polarity * plasma-membrane-protein * cultured tobacco cells * arabidopsis-thaliana * gravitropic response * brefeldin-a * Plant hormones * Transport * Inhibitors * Auxin * Cytokinins * Strigolactones * Abscisic acid * Cell biology Subject RIV: ED - Physiology Impact factor: 2.870, year: 2016

  8. Transporter Protein-Coupled DPCPX Nanoconjugates Induce Diaphragmatic Recovery after SCI by Blocking Adenosine A1 Receptors.

    Science.gov (United States)

    Minic, Zeljka; Zhang, Yanhua; Mao, Guangzhao; Goshgarian, Harry G

    2016-03-23

    Respiratory complications in patients with spinal cord injury (SCI) are common and have a negative impact on the quality of patients' lives. Systemic administration of drugs that improve respiratory function often cause deleterious side effects. The present study examines the applicability of a novel nanotechnology-based drug delivery system, which induces recovery of diaphragm function after SCI in the adult rat model. We developed a protein-coupled nanoconjugate to selectively deliver by transsynaptic transport small therapeutic amounts of an A1 adenosine receptor antagonist to the respiratory centers. A single administration of the nanoconjugate restored 75% of the respiratory drive at 0.1% of the systemic therapeutic drug dose. The reduction of the systemic dose may obviate the side effects. The recovery lasted for 4 weeks (the longest period studied). These findings have translational implications for patients with respiratory dysfunction after SCI. The leading causes of death in humans following SCI are respiratory complications secondary to paralysis of respiratory muscles. Systemic administration of methylxantines improves respiratory function but also leads to the development of deleterious side effects due to actions of the drug on nonrespiratory sites. The importance of the present study lies in the novel drug delivery approach that uses nanotechnology to selectively deliver recovery-inducing drugs to the respiratory centers exclusively. This strategy allows for a reduction in the therapeutic drug dose, which may reduce harmful side effects and markedly improve the quality of life for SCI patients. Copyright © 2016 the authors 0270-6474/16/363441-12$15.00/0.

  9. Small Molecule Membrane Transporters in the Mammalian Podocyte: A Pathogenic and Therapeutic Target

    Directory of Open Access Journals (Sweden)

    Cristina Zennaro

    2014-11-01

    Full Text Available The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development.

  10. A 'periodic table' for protein structures.

    Science.gov (United States)

    Taylor, William R

    2002-04-11

    Current structural genomics programs aim systematically to determine the structures of all proteins coded in both human and other genomes, providing a complete picture of the number and variety of protein structures that exist. In the past, estimates have been made on the basis of the incomplete sample of structures currently known. These estimates have varied greatly (between 1,000 and 10,000; see for example refs 1 and 2), partly because of limited sample size but also owing to the difficulties of distinguishing one structure from another. This distinction is usually topological, based on the fold of the protein; however, in strict topological terms (neglecting to consider intra-chain cross-links), protein chains are open strings and hence are all identical. To avoid this trivial result, topologies are determined by considering secondary links in the form of intra-chain hydrogen bonds (secondary structure) and tertiary links formed by the packing of secondary structures. However, small additions to or loss of structure can make large changes to these perceived topologies and such subjective solutions are neither robust nor amenable to automation. Here I formalize both secondary and tertiary links to allow the rigorous and automatic definition of protein topology.

  11. Characteristics of the Secondary Divertor on DIII-D

    Science.gov (United States)

    Watkins, J. G.; Lasnier, C. J.; Leonard, A. W.; Evans, T. E.; Pitts, R.; Stangeby, P. C.; Boedo, J. A.; Moyer, R. A.; Rudakov, D. L.

    2009-11-01

    In order to address a concern that the ITER secondary divertor strike plates may be insufficiently robust to handle the incident pulses of particles and energy from ELMs, we performed dedicated studies of the secondary divertor plasma and scrape-off layer (SOL). Detailed measurements of the ELM energy and particle deposition footprint on the secondary divertor target plates were made with a fast IR camera and Langmuir probes and SOL profile and transport measurements were made with reciprocating probes. The secondary divertor and SOL conditions depended on changes in the magnetic balance and the core plasma density. Larger density resulted in smaller ELMs and the magnetic balance affected how many ELM particles coupled to the secondary SOL and divertor. Particularly striking are the images from a new fast IR camera that resolve ELM heat pulses and show spiral patterns with multiple peaks during ELMs in the secondary divertor.

  12. Bioinformatic survey of ABC transporters in dermatophytes.

    Science.gov (United States)

    Gadzalski, Marek; Ciesielska, Anita; Stączek, Paweł

    2016-01-15

    ATP binding cassette (ABC) transporters constitute a very large and ubiquitous superfamily of membrane proteins. They are responsible for ATP hydrolysis driven translocation of countless substrates. Being a very old and diverse group of proteins present in all organisms they share a common feature, which is the presence of an evolutionary conservative nucleotide binding domain (NBD)--the engine that drives the transport. Another common domain is a transmembrane domain (TMD) which consists of several membrane-spanning helices. This part of protein is substrate-specific, thus it is much more variable. ABC transporters are known for driving drug efflux in many pathogens and cancer cells, therefore they are the subject of extensive studies. There are many examples of conferring a drug resistance phenotype in fungal pathogens by ABC transporters, however, little is known about these proteins in dermatophytes--a group of fungi causing superficial mycoses. So far only a single ABC transporter has been extensively studied in this group of pathogens. We analyzed available genomic sequences of seven dermatophyte species in order to provide an insight into dermatophyte ABC protein inventory. Phylogenetic studies of ABC transporter genes and their products were conducted and included ABC transporters of other fungi. Our results show that each dermatophyte genome studied possesses a great variety of ABC transporter genes. Detailed analysis of selected genes and their products indicates that relatively recent duplication of ABC transporter genes could lead to novel substrate specificity. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. The expression and function of fatty acid transport protein-2 and -4 in the murine placenta.

    Directory of Open Access Journals (Sweden)

    Takuya Mishima

    Full Text Available The uptake and trans-placental trafficking of fatty acids from the maternal blood into the fetal circulation are essential for embryonic development, and involve several families of proteins. Fatty acid transport proteins (FATPs uniquely transport fatty acids into cells. We surmised that placental FATPs are germane for fetal growth, and are regulated during hypoxic stress, which is associated with reduced fat supply to the fetus.Using cultured primary term human trophoblasts we found that FATP2, FATP4 and FATP6 were highly expressed in trophoblasts. Hypoxia enhanced the expression of trophoblastic FATP2 and reduced the expression of FATP4, with no change in FATP6. We also found that Fatp2 and Fatp4 are expressed in the mouse amnion and placenta, respectively. Mice deficient in Fatp2 or Fatp4 did not deviate from normal Mendelian distribution, with both embryos and placentas exhibiting normal weight and morphology, triglyceride content, and expression of genes related to fatty acid mobilization.We conclude that even though hypoxia regulates the expression of FATP2 and FATP4 in human trophoblasts, mouse Fatp2 and Fatp4 are not essential for intrauterine fetal growth.

  14. Regional distribution of serotonin transporter protein in postmortem human brain

    International Nuclear Information System (INIS)

    Kish, Stephen J.; Furukawa, Yoshiaki; Chang Lijan; Tong Junchao; Ginovart, Nathalie; Wilson, Alan; Houle, Sylvain; Meyer, Jeffrey H.

    2005-01-01

    Introduction: The primary approach in assessing the status of brain serotonin neurons in human conditions such as major depression and exposure to the illicit drug ecstasy has been the use of neuroimaging procedures involving radiotracers that bind to the serotonin transporter (SERT). However, there has been no consistency in the selection of a 'SERT-free' reference region for the estimation of free and nonspecific binding, as occipital cortex, cerebellum and white matter have all been employed. Objective and Methods: To identify areas of human brain that might have very low SERT levels, we measured, by a semiquantitative Western blotting procedure, SERT protein immunoreactivity throughout the postmortem brain of seven normal adult subjects. Results: Serotonin transporter could be quantitated in all examined brain areas. However, the SERT concentration in cerebellar cortex and white matter were only at trace values, being approximately 20% of average cerebral cortex and 5% of average striatum values. Conclusion: Although none of the examined brain areas are completely free of SERT, human cerebellar cortex has low SERT binding as compared to other examined brain regions, with the exception of white matter. Since the cerebellar cortical SERT binding is not zero, this region will not be a suitable reference region for SERT radioligands with very low free and nonspecific binding. For SERT radioligands with reasonably high free and nonspecific binding, the cerebellar cortex should be a useful reference region, provided other necessary radioligand assumptions are met

  15. Regional distribution of serotonin transporter protein in postmortem human brain

    Energy Technology Data Exchange (ETDEWEB)

    Kish, Stephen J. [Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8 (Canada)]. E-mail: Stephen_Kish@CAMH.net; Furukawa, Yoshiaki [Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8 (Canada); Chang Lijan [Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8 (Canada); Tong Junchao [Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8 (Canada); Ginovart, Nathalie [PET Centre, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8 (Canada); Wilson, Alan [PET Centre, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8 (Canada); Houle, Sylvain [PET Centre, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8 (Canada); Meyer, Jeffrey H. [PET Centre, Centre for Addiction and Mental Health, Toronto, ON, M5T 1R8 (Canada)

    2005-02-01

    Introduction: The primary approach in assessing the status of brain serotonin neurons in human conditions such as major depression and exposure to the illicit drug ecstasy has been the use of neuroimaging procedures involving radiotracers that bind to the serotonin transporter (SERT). However, there has been no consistency in the selection of a 'SERT-free' reference region for the estimation of free and nonspecific binding, as occipital cortex, cerebellum and white matter have all been employed. Objective and Methods: To identify areas of human brain that might have very low SERT levels, we measured, by a semiquantitative Western blotting procedure, SERT protein immunoreactivity throughout the postmortem brain of seven normal adult subjects. Results: Serotonin transporter could be quantitated in all examined brain areas. However, the SERT concentration in cerebellar cortex and white matter were only at trace values, being approximately 20% of average cerebral cortex and 5% of average striatum values. Conclusion: Although none of the examined brain areas are completely free of SERT, human cerebellar cortex has low SERT binding as compared to other examined brain regions, with the exception of white matter. Since the cerebellar cortical SERT binding is not zero, this region will not be a suitable reference region for SERT radioligands with very low free and nonspecific binding. For SERT radioligands with reasonably high free and nonspecific binding, the cerebellar cortex should be a useful reference region, provided other necessary radioligand assumptions are met.

  16. Plant Transporter Identification

    DEFF Research Database (Denmark)

    Larsen, Bo

    Membrane transport proteins (transporters) play a critical role for numerous biological processes, by controlling the movements of ions and molecules in and out of cells. In plants, transporters thus function as gatekeepers between the plant and its surrounding environment and between organs......, tissues, cells and intracellular compartments. Since plants are highly compartmentalized organisms with complex transportation infrastructures, they consequently have many transporters. However, the vast majority of predicted transporters have not yet been experimentally verified to have transport...... activity. This project contains a review of the implemented methods, which have led to plant transporter identification, and present our progress on creating a high-throughput functional genomics transporter identification platform....

  17. FT-IR spectroscopic studies of protein secondary structures for breast cancer diagnosis

    International Nuclear Information System (INIS)

    Karamancheva, I; Simonova, D.; Milev, A.

    2013-01-01

    Full text: Roughly 14 million new cancer cases and 8 million cancer deaths have occurred worldwide in 2012. At least 30 % of all cancer cases and 40 % of the cancer deaths should be avoided by improving the early detection. Fourier transform infrared (FT-IR) spectroscopy has shown many advantages as a tool for the detection of cancer over the traditional methods such as histopathological analysis, X-ray transmission, ultrasonic and computer tomography techniques. With the aim to establish the FT-IR spectroscopy as an alternative method for the diagnosis of human cancers, we have made several studies to examine in details the spectroscopic properties of normal and carcinomatous tissues. Human breast tissues were obtained immediately after surgical breast resection with the informed patient's consent. In our studies we made extensive use of Fourier self-deconvolution, second-order derivatization, difference spectra, curve-fitting procedures and quantitative determinations according to Beer's law. Cancer is a multi-step process. Characteristic differences in both the frequencies and the intensity ratios of several bands have been revealed. Considerable differences have been found in the spectral patterns. The most important and informative region in the mid-IR for determination of protein secondary structure is the amide I and amide II region. The bands between 1730 and 1600 cm -1 are highly sensitive to conformational changes. Considerable changes were observed in the A1735/A1652 absorbance ratio, which provides a measure for the content of a- helix and P-sheet domains. Our investigations have shown that the major biomarker peaks are in the amide I and amide II regions. In the so called 'fingerprint region' many molecular constituents such as lipids, phospholipids, proteins, DNA and RNA, carbohydrates and metabolites may overlap and the quantitative interpretation is impossible. The spectrum may therefore reflect only the average biochemical composition.; key words

  18. ETRAN, Electron Transport and Gamma Transport with Secondary Radiation in Slab by Monte-Carlo

    International Nuclear Information System (INIS)

    1992-01-01

    A - Nature of physical problem solved: ETRAN computes the transport of electrons and photons through plane-parallel slab targets that have a finite thickness in one dimension and are unbound in the other two-dimensions. The incident radiation can consist of a beam of either electrons or photons with specified spectral and directional distribution. Options are available by which all orders of the electron-photon cascade can be included in the calculation. Thus electrons are allowed to give rise to secondary knock-on electrons, continuous Bremsstrahlung and characteristic x-rays; and photons are allowed to produce photo-electrons, Compton electrons, and electron- positron pairs. Annihilation quanta, fluorescence radiation, and Auger electrons are also taken into account. If desired, the Monte- Carlo histories of all generations of secondary radiations are followed. The information produced by ETRAN includes the following items: 1) reflection and transmission of electrons or photons, differential in energy and direction; 2) the production of continuous Bremsstrahlung and characteristic x-rays by electrons and the emergence of such radiations from the target (differential in photon energy and direction); 3) the spectrum of the amounts of energy left behind in a thick target by an incident electron beam; 4) the deposition of energy and charge by an electron beam as function of the depth in the target; 5) the flux of electrons, differential in energy, as function of the depth in the target. B - Method of solution: A programme called DATAPAC-4 takes data for a particular material from a library tape and further processes them. The function of DATAPAC-4 is to produce single-scattering and multiple-scattering data in the form of tabular arrays (again stored on magnetic tape) which facilitate the rapid sampling of electron and photon Monte Carlo histories in ETRAN. The photon component of the electron-photon cascade is calculated by conventional random sampling that imitates

  19. Effect of colchicine on the transport of precursor enamel protein in secretory ameloblasts studied by 3H-proline radioautography in vitro

    International Nuclear Information System (INIS)

    Matsuo, S.; Takano, Y.; Wakisaka, S.; Ichikawa, H.; Nishikawa, S.; Akai, M.

    1988-01-01

    The incorporation of 3H-proline into the secretory ameloblasts of rat molar tooth germs cultured with or without colchicine was studied by light and electron microscope radioautography to determine the function of microtubules in the transport of precursor enamel protein from the rough-surfaced endoplasmic reticulum (rER) to the Golgi cisternae. The grain counts over the transitional vesicles, which accumulated in various cellular regions with colchicine treatment, continued to increase with chase time, unlike in controls. At 30 and 90 min chase, these counts were significantly higher than in controls. Moreover, the total grain count over the organelles (rER, pale granules, and transitional vesicles), which are positioned before the Golgi cisternae in the synthetic pathway, maintained a significantly higher level at 90 min chase in colchicine-treated tooth germs than in controls. The transport of synthesized protein to the Golgi cisternae via transitional vesicles was suppressed in colchicine-treated tooth germs. Some grains appeared with time over pale granular materials that appeared in the intercellular spaces of secretory ameloblasts with colchicine treatment. However, at each chase period, the grain count over pale granular materials was not so high as the count over the enamel in control. The present results indicate that colchicine affects the transport of newly synthesized protein from the rER to the Golgi cisterna via transitional vesicles, probably by interfering with the oriented transport related to microtubular function. It is suggested that the microtubular system may be concerned with the movement of the transitional vesicles

  20. A Reference Database for Circular Dichroism Spectroscopy Covering Fold and Secondary Structure Space

    International Nuclear Information System (INIS)

    Lees, J.; Miles, A.; Wien, F.; Wallace, B.

    2006-01-01

    Circular Dichroism (CD) spectroscopy is a long-established technique for studying protein secondary structures in solution. Empirical analyses of CD data rely on the availability of reference datasets comprised of far-UV CD spectra of proteins whose crystal structures have been determined. This article reports on the creation of a new reference dataset which effectively covers both secondary structure and fold space, and uses the higher information content available in synchrotron radiation circular dichroism (SRCD) spectra to more accurately predict secondary structure than has been possible with existing reference datasets. It also examines the effects of wavelength range, structural redundancy and different means of categorizing secondary structures on the accuracy of the analyses. In addition, it describes a novel use of hierarchical cluster analyses to identify protein relatedness based on spectral properties alone. The databases are shown to be applicable in both conventional CD and SRCD spectroscopic analyses of proteins. Hence, by combining new bioinformatics and biophysical methods, a database has been produced that should have wide applicability as a tool for structural molecular biology

  1. Membrane Transporters: Structure, Function and Targets for Drug Design

    Science.gov (United States)

    Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

    Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

  2. Modularity in protein structures: study on all-alpha proteins.

    Science.gov (United States)

    Khan, Taushif; Ghosh, Indira

    2015-01-01

    Modularity is known as one of the most important features of protein's robust and efficient design. The architecture and topology of proteins play a vital role by providing necessary robust scaffolds to support organism's growth and survival in constant evolutionary pressure. These complex biomolecules can be represented by several layers of modular architecture, but it is pivotal to understand and explore the smallest biologically relevant structural component. In the present study, we have developed a component-based method, using protein's secondary structures and their arrangements (i.e. patterns) in order to investigate its structural space. Our result on all-alpha protein shows that the known structural space is highly populated with limited set of structural patterns. We have also noticed that these frequently observed structural patterns are present as modules or "building blocks" in large proteins (i.e. higher secondary structure content). From structural descriptor analysis, observed patterns are found to be within similar deviation; however, frequent patterns are found to be distinctly occurring in diverse functions e.g. in enzymatic classes and reactions. In this study, we are introducing a simple approach to explore protein structural space using combinatorial- and graph-based geometry methods, which can be used to describe modularity in protein structures. Moreover, analysis indicates that protein function seems to be the driving force that shapes the known structure space.

  3. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C., E-mail: cdirusso2@unl.edu

    2015-09-25

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC{sub 50} 8–11 μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC{sub 50} 58 μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of {sup 13}C-oleate demonstrating its potential as a therapeutic agent. - Highlights: • Grassofermata is a small compound inhibitor of FATP2. • Uptake inhibition is specific for long chain fatty acids. • Uptake kinetics shows low specificity for adipocytes compared to other cell types. • Inhibition is by a non-competitive mechanism. • Atypical antipsychotics do not inhibit FA uptake by comparison with Grassofermata.

  4. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    International Nuclear Information System (INIS)

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-01-01

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC 50 8–11 μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC 50 58 μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of 13 C-oleate demonstrating its potential as a therapeutic agent. - Highlights: • Grassofermata is a small compound inhibitor of FATP2. • Uptake inhibition is specific for long chain fatty acids. • Uptake kinetics shows low specificity for adipocytes compared to other cell types. • Inhibition is by a non-competitive mechanism. • Atypical antipsychotics do not inhibit FA uptake by comparison with Grassofermata

  5. Systematization of the protein sequence diversity in enzymes related to secondary metabolic pathways in plants, in the context of big data biology inspired by the KNApSAcK motorcycle database.

    Science.gov (United States)

    Ikeda, Shun; Abe, Takashi; Nakamura, Yukiko; Kibinge, Nelson; Hirai Morita, Aki; Nakatani, Atsushi; Ono, Naoaki; Ikemura, Toshimichi; Nakamura, Kensuke; Altaf-Ul-Amin, Md; Kanaya, Shigehiko

    2013-05-01

    Biology is increasingly becoming a data-intensive science with the recent progress of the omics fields, e.g. genomics, transcriptomics, proteomics and metabolomics. The species-metabolite relationship database, KNApSAcK Core, has been widely utilized and cited in metabolomics research, and chronological analysis of that research work has helped to reveal recent trends in metabolomics research. To meet the needs of these trends, the KNApSAcK database has been extended by incorporating a secondary metabolic pathway database called Motorcycle DB. We examined the enzyme sequence diversity related to secondary metabolism by means of batch-learning self-organizing maps (BL-SOMs). Initially, we constructed a map by using a big data matrix consisting of the frequencies of all possible dipeptides in the protein sequence segments of plants and bacteria. The enzyme sequence diversity of the secondary metabolic pathways was examined by identifying clusters of segments associated with certain enzyme groups in the resulting map. The extent of diversity of 15 secondary metabolic enzyme groups is discussed. Data-intensive approaches such as BL-SOM applied to big data matrices are needed for systematizing protein sequences. Handling big data has become an inevitable part of biology.

  6. Solute carrier transporters: potential targets for digestive system neoplasms.

    Science.gov (United States)

    Xie, Jing; Zhu, Xiao Yan; Liu, Lu Ming; Meng, Zhi Qiang

    2018-01-01

    Digestive system neoplasms are the leading causes of cancer-related death all over the world. Solute carrier (SLC) superfamily is composed of a series of transporters that are ubiquitously expressed in organs and tissues of digestive systems and mediate specific uptake of small molecule substrates in facilitative manner. Given the important role of SLC proteins in maintaining normal functions of digestive system, dysregulation of these protein in digestive system neoplasms may deliver biological and clinical significance that deserves systemic studies. In this review, we critically summarized the recent advances in understanding the role of SLC proteins in digestive system neoplasms. We highlighted that several SLC subfamilies, including metal ion transporters, transporters of glucose and other sugars, transporters of urea, neurotransmitters and biogenic amines, ammonium and choline, inorganic cation/anion transporters, transporters of nucleotide, amino acid and oligopeptide organic anion transporters, transporters of vitamins and cofactors and mitochondrial carrier, may play important roles in mediating the initiation, progression, metastasis, and chemoresistance of digestive system neoplasms. Proteins in these SLC subfamilies may also have diagnostic and prognostic values to particular cancer types. Differential expression of SLC proteins in tumors of digestive system was analyzed by extracting data from human cancer database, which revealed that the roles of SLC proteins may either be dependent on the substrates they transport or be tissue specific. In addition, small molecule modulators that pharmacologically regulate the functions of SLC proteins were discussed for their possible application in the treatment of digestive system neoplasms. This review highlighted the potential of SLC family proteins as drug target for the treatment of digestive system neoplasms.

  7. Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons.

    Science.gov (United States)

    Niwa, Shinsuke

    2017-10-18

    Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

  8. Characteristics of Mammalian Rh Glycoproteins (SLC42 transporters) and Their Role in Acid-Base Transport

    Science.gov (United States)

    Nakhoul, Nazih L.; Hamm, L. Lee

    2012-01-01

    The mammalian Rh glycoproteins belong to the solute transporter family SLC42 and include RhAG, present in red blood cells, and two non-erythroid members RhBG and RhCG that are expressed in various tissues, including kidney, liver, skin and the GI tract. The Rh proteins in the red blood cell form an “Rh complex” made up of one D-subunit, one CE-subunit and two RhAG subunits. The Rh complex has a well-known antigenic effect but also contributes to the stability of the red cell membrane. RhBG and RhCG are related to the NH4+ transporters of the yeast and bacteria but their exact function is yet to be determined. This review describes the expression and molecular properties of these membrane proteins and their potential role as NH3/NH4+ and CO2 transporters. The likelihood that these proteins transport gases such as CO2 or NH3 is novel and significant. The review also describes the physiological importance of these proteins and their relevance to human disease. PMID:23506896

  9. The expression and regulation of glucose transporters in tumor cells

    Directory of Open Access Journals (Sweden)

    Pengfei Zhao

    2016-12-01

    Full Text Available Glucose transporter proteins are involved in many physiological and biochemical processes. In particular, the high expressions of sodium-glucose cotransporter and glucose transporter proteins in tumor cells show that these two transporters play a key role in tumor cell metabolism. Studying the crystal structure and conformation of human glucose transporter proteins has enabled the development of drugs based on specific binding sites, opening up a new path towards more effective cancer treatments. This mini review serves to summarize our existing understanding of the metabolic pathways of tumor cells, focusing on the roles of glucose transporter proteins.

  10. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  11. Is air transport of stroke patients faster than ground transport?

    DEFF Research Database (Denmark)

    Hesselfeldt, Rasmus; Gyllenborg, Jesper; Steinmetz, Jacob

    2014-01-01

    BACKGROUND: Helicopters are widely used for interhospital transfers of stroke patients, but the benefit is sparsely documented. We hypothesised that helicopter transport would reduce system delay to thrombolytic treatment at the regional stroke centre. METHODS: In this prospective controlled...... observational study, we included patients referred to a stroke centre if their ground transport time exceeded 30 min, or they were transported by a secondarily dispatched, physician-staffed helicopter. The primary endpoint was time from telephone contact to triaging neurologist to arrival in the stroke centre....... Secondary endpoints included modified Rankin Scale at 3 months, 30-day and 1-year mortality. RESULTS: A total of 330 patients were included; 265 with ground transport and 65 with helicopter, of which 87 (33%) and 22 (34%), received thrombolysis, respectively (p=0.88). Time from contact to triaging...

  12. Specific serum protein changes associated with primary and secondary Strongylus vulgaris infections in pony yearlings.

    Science.gov (United States)

    Kent, J E

    1987-03-01

    The concentrations of haptoglobin, immunoglobin (Ig)G(T) and IgG were measured in the serum of four previously parasite-free pony yearlings following a single dose of 700 (Group H) or 200 (Group L) stage three Strongylus vulgaris larvae (L3) and following a reinfection with the same doses 34 weeks later. The results are compared with an uninfected control pony. The haptoglobin concentration increased during Weeks 1 to 6 and 14 to 17 after infection in the serum of the ponies receiving 200 L3, but in only one pony dosed with 700 L3 (during Weeks 1 to 16). The serum haptoglobin also increased during the first seven weeks after the second infection, in three of the four ponies following the second dose of larvae. The serum IgG(T) concentration started to increase from Week 6 or 9 in the ponies given 700 L3, reaching peaks of 44 and 32 g/litre respectively, eight to nine weeks later, compared with a peak of 16 g/litre 20 to 22 weeks after infection in ponies dosed with 200 L3. The IgG(T) concentration increased to a maximum of 25 g/litre in the serum of only one of the four ponies after the reinfection. The serum IgG concentration in all ponies increased nearly twofold during the first eight weeks after both the primary and secondary dose of larvae. It is concluded that the measurement of specific proteins is more reliable and quicker than the electrophoretic separation and quantitation of protein bands, in tracing changes in serum proteins following the artificial infection of ponies with S vulgaris larvae.

  13. Glucose Elevates NITRATE TRANSPORTER2.1 Protein Levels and Nitrate Transport Activity Independently of Its HEXOKINASE1-Mediated Stimulation of NITRATE TRANSPORTER2.1 Expression1[W][OPEN

    Science.gov (United States)

    de Jong, Femke; Thodey, Kate; Lejay, Laurence V.; Bevan, Michael W.

    2014-01-01

    Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth. PMID:24272701

  14. Protein malnutrition blunts the increment of taurine transporter expression by a high-fat diet and impairs taurine reestablishment of insulin secretion.

    Science.gov (United States)

    Branco, Renato Chaves Souto; Camargo, Rafael Ludemann; Batista, Thiago Martins; Vettorazzi, Jean Franciesco; Borck, Patrícia Cristine; Dos Santos-Silva, Junia Carolina Rebelo; Boschero, Antonio Carlos; Zoppi, Cláudio Cesar; Carneiro, Everardo Magalhães

    2017-09-01

    Taurine (Tau) restores β-cell function in obesity; however, its action is lost in malnourished obese rodents. Here, we investigated the mechanisms involved in the lack of effects of Tau in this model. C57BL/6 mice were fed a control diet (CD) (14% protein) or a protein-restricted diet (RD) (6% protein) for 6 wk. Afterward, mice received a high-fat diet (HFD) for 8 wk [CD + HFD (CH) and RD + HFD (RH)] with or without 5% Tau supplementation after weaning on their drinking water [CH + Tau (CHT) and RH + Tau (RHT)]. The HFD increased insulin secretion through mitochondrial metabolism in CH and RH. Tau prevented all those alterations in CHT only. The expression of the taurine transporter (Tau-T), as well as Tau content in pancreatic islets, was increased in CH but had no effect on RH. Protein malnutrition programs β cells and impairs Tau-induced restoration of mitochondrial metabolism and biogenesis. This may be associated with modulation of the expression of Tau-T in pancreatic islets, which may be responsible for the absence of effect of Tau in protein-malnourished obese mice.-Branco, R. C. S., Camargo, R. L., Batista, T. M., Vettorazzi, J. F., Borck, P. C., dos Santos-Silva, J. C. R., Boschero, A. C., Zoppi, C. C., Carneiro, E. M. Protein malnutrition blunts the increment of taurine transporter expression by a high-fat diet and impairs taurine reestablishment of insulin secretion. © FASEB.

  15. A nu-space for ICS: characterization and application to measure protein transport in live cells.

    Science.gov (United States)

    Potvin-Trottier, Laurent; Chen, Lingfeng; Horwitz, Alan Rick; Wiseman, Paul W

    2013-08-01

    We introduce a new generalized theoretical framework for image correlation spectroscopy (ICS). Using this framework, we extend the ICS method in time-frequency ( ν , nu) space to map molecular flow of fluorescently tagged proteins in individual living cells. Even in the presence of a dominant immobile population of fluorescent molecules, nu-space ICS (nICS) provides an unbiased velocity measurement, as well as the diffusion coefficient of the flow, without requiring filtering. We also develop and characterize a tunable frequency-filter for STICS that allows quantification of the density, the diffusion coefficient and the velocity of biased diffusion. We show that the techniques are accurate over a wide range of parameter space in computer simulation. We then characterize the retrograde flow of adhesion proteins ( α 6- and αLβ 2-GFP integrins and mCherry-paxillin) in CHO.B2 cells plated on laminin and ICAM ligands respectively. STICS with a tunable frequency filter, in conjunction with nICS, measures two new transport parameters, the density and transport bias coefficient (a measure of the diffusive character of a flow/biased diffusion), showing that molecular flow in this cell system has a significant diffusive component. Our results suggest that the integrinligand interaction, along with the internal myosin-motor generated force, varies for different integrin-ligand pairs, consistent with previous results.

  16. Preventing secondary cases of invasive meningococcal capsular group B (MenB) disease using a recently-licensed, multi-component, protein-based vaccine (Bexsero(®)).

    Science.gov (United States)

    Ladhani, Shamez N; Cordery, Rebecca; Mandal, Sema; Christensen, Hannah; Campbell, Helen; Borrow, Ray; Ramsay, Mary E

    2014-11-01

    To assess the potential use of a protein-based meningococcal group B (MenB) vaccine (Bexsero(®)) in addition to antibiotic chemoprophylaxis for preventing secondary cases. Published studies on the risk of secondary meningococcal infections were used to estimate the numbers needed to vaccinate (NNV) with Bexsero(®) to prevent a secondary case in household and educational settings. Most secondary cases occur within a few days of diagnosis in the index case. Unlike conjugate vaccines, early protection offered after a single dose of Bexsero(®) is likely to be low, particularly in young children, who are at higher risk of secondary infection. NNV was dependent on predicted meningococcal strain coverage, estimated onset of protection after one Bexsero(®) dose and estimated vaccine efficacy. Even in the most favourable scenario where we assume the vaccine is administered within 4 days of the index case and prevents 90% of cases occurring after 14 days, the NNV for household contacts was >1000. NNV in educational settings was much higher. The estimated NNV should be taken into account when deciding policy to recommend Bexsero(®) for close contacts of single cases in household or educational settings. Bexsero(®) may have a protective role in clusters and outbreaks. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  17. Systemic transport of Alfalfa mosaic virus can be mediated by the movement proteins of several viruses assigned to five genera of the 30K family.

    Science.gov (United States)

    Fajardo, Thor V M; Peiró, Ana; Pallás, Vicente; Sánchez-Navarro, Jesús

    2013-03-01

    We previously showed that the movement protein (MP) gene of Alfalfa mosaic virus (AMV) is functionally exchangeable for the cell-to-cell transport of the corresponding genes of Tobacco mosaic virus (TMV), Brome mosaic virus, Prunus necrotic ringspot virus, Cucumber mosaic virus and Cowpea mosaic virus. We have analysed the capacity of the heterologous MPs to systemically transport the corresponding chimeric AMV genome. All MPs were competent in systemic transport but required the fusion at their C terminus of the coat protein-interacting C-terminal 44 aa (A44) of the AMV MP. Except for the TMV MP, the presence of the hybrid virus in upper leaves correlated with the capacity to move locally. These results suggest that all the MPs assigned to the 30K superfamily should be exchangeable not only for local virus movement but also for systemic transport when the A44 fragment is present.

  18. Multiscale Roughness Influencing on Transport Behavior of Passive Solute through a Single Self-affine Fracture

    Science.gov (United States)

    Dou, Z.

    2017-12-01

    In this study, the influence of multi-scale roughness on transport behavior of the passive solute through the self-affine fracture was investigated. The single self-affine fracture was constructed by the successive random additions (SRA) and the fracture roughness was decomposed into two different scales (i.e. large-scale primary roughness and small-scale secondary roughness) by the Wavelet analysis technique. The fluid flow in fractures, which was characterized by the Forchheimer's law, showed the non-linear flow behaviors such as eddies and tortuous streamlines. The results indicated that the small-scale secondary roughness was primarily responsible for the non-linear flow behaviors. The direct simulations of asymptotic passive solute transport represented the Non-Fickian transport characteristics (i.e. early arrivals and long tails) in breakthrough curves (BTCs) and residence time distributions (RTDs) with and without consideration of the secondary roughness. Analysis of multiscale BTCs and RTDs showed that the small-scale secondary roughness played a significant role in enhancing the Non-Fickian transport characteristics. We found that removing small-scale secondary roughness led to the lengthening arrival and shortening tail. The peak concentration in BTCs decreased as the secondary roughness was removed, implying that the secondary could also enhance the solute dilution. The estimated BTCs by the Fickian advection-dispersion equation (ADE) yielded errors which decreased with the small-scale secondary roughness being removed. The mobile-immobile model (MIM) was alternatively implemented to characterize the Non-Fickian transport. We found that the MIM was more capable of estimating Non-Fickian BTCs. The small-scale secondary roughness resulted in the decreasing mobile domain fraction and the increasing mass exchange rate between immobile and mobile domains. The estimated parameters from the MIM could provide insight into the inherent mechanism of roughness

  19. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either by facilitat...... describes the steps in the analysis of the secondary structure of the pre-mRNA and its possible relation to splicing. As a working example, we use the case of yeast and the problem of the recognition of the 3' splice site (3'ss).......In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either...

  20. 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

  1. Physicochemical Properties of Whey-Protein-Stabilized Astaxanthin Nanodispersion and Its Transport via a Caco-2 Monolayer.

    Science.gov (United States)

    Shen, Xue; Zhao, Changhui; Lu, Jing; Guo, Mingruo

    2018-02-14

    Astaxanthin nanodispersion was prepared using whey protein isolate (WPI) and polymerized whey protein (PWP) through an emulsification-evaporation technique. The physicochemical properties of the astaxanthin nanodispersion were evaluated, and the transport of astaxanthin was assessed using a Caco-2 cell monolayer model. The astaxanthin nanodispersions stabilized by WPI and PWP (2.5%, w/w) had a small particle size (121 ± 4.9 and 80.4 ± 5.9 nm, respectively), negative ζ potential (-19.3 ± 1.5 and -35.0 ± 2.2 mV, respectively), and high encapsulation efficiency (92.1 ± 2.9 and 93.5 ± 2.4%, respectively). Differential scanning calorimetry curves indicated that amorphous astaxanthin existed in both astaxanthin nanodispersions. Whey-protein-stabilized astaxanthin nanodispersion showed resistance to pepsin digestion but readily released astaxanthin after trypsin digestion. The nanodispersions showed no cytotoxicity to Caco-2 cells at a protein concentration below 10 mg/mL. WPI- and PWP-stabilized nanodispersions improved the apparent permeability coefficient (P app ) of Caco-2 cells to astaxanthin by 10.3- and 16.1-fold, respectively. The results indicated that whey-protein-stabilized nanodispersion is a good vehicle to deliver lipophilic bioactive compounds, such as astaxanthin, and to improve their bioavailability.

  2. Brittle stalk 2 encodes a putative glycosylphosphatidylinositol-anchored protein that affects mechanical strength of maize tissues by altering the composition and structure of secondary cell walls.

    Science.gov (United States)

    Ching, Ada; Dhugga, Kanwarpal S; Appenzeller, Laura; Meeley, Robert; Bourett, Timothy M; Howard, Richard J; Rafalski, Antoni

    2006-10-01

    A spontaneous maize mutant, brittle stalk-2 (bk2-ref), exhibits dramatically reduced tissue mechanical strength. Reduction in mechanical strength in the stalk tissue was highly correlated with a reduction in the amount of cellulose and an uneven deposition of secondary cell wall material in the subepidermal and perivascular sclerenchyma fibers. Cell wall accounted for two-thirds of the observed reduction in dry matter content per unit length of the mutant stalk in comparison to the wildtype stalk. Although the cell wall composition was significantly altered in the mutant in comparison to the wildtype stalks, no compensation by lignin and cell wall matrix for reduced cellulose amount was observed. We demonstrate that Bk2 encodes a Cobra-like protein that is homologous to the rice Bc1 protein. In the bk2-ref gene, a 1 kb transposon-like element is inserted in the beginning of the second exon, disrupting the open reading frame. The Bk2 gene was expressed in the stalk, husk, root, and leaf tissues, but not in the embryo, endosperm, pollen, silk, or other tissues with comparatively few or no secondary cell wall containing cells. The highest expression was in the isolated vascular bundles. In agreement with its role in secondary wall formation, the expression pattern of the Bk2 gene was very similar to that of the ZmCesA10, ZmCesA11, and ZmCesA12 genes, which are known to be involved in secondary wall formation. We have isolated an independent Mutator-tagged allele of bk2, referred to as bk2-Mu7, the phenotype of which is similar to that of the spontaneous mutant. Our results demonstrate that mutations in the Bk2 gene affect stalk strength in maize by interfering with the deposition of cellulose in the secondary cell wall in fiber cells.

  3. Placental Expression of Glucose Transporter Proteins in Pregnancies Complicated by Gestational and Pregestational Diabetes Mellitus.

    Science.gov (United States)

    Stanirowski, Paweł Jan; Szukiewicz, Dariusz; Pazura-Turowska, Monika; Sawicki, Włodzimierz; Cendrowski, Krzysztof

    2018-04-01

    Gestational diabetes mellitus and pregestational diabetes mellitus constitute carbohydrate metabolism disorders, which, if not diagnosed and adequately treated, lead to serious and often life-threatening pregnancy complications. According to a recently formulated hypothesis, some diabetes-related complications, such as fetal macrosomia, may be the result of disturbances in the transplacental transport of nutrients-in particular, excessive maternal-fetal glucose transfer. Throughout pregnancy, glucose flux across the placenta is mediated by the group of facilitative glucose transporters (GLUT), the expression of which in different placental compartments is the precondition for effective glucose uptake from maternal blood and its subsequent transfer to the fetal circulation. In diabetes-complicated pregnancies, the location, expression and activity of glucose transporters are modified to an extent that results in alterations in the maternal-fetal glucose exchange, potentially leading to an excessive supply of energy substrates to the fetus. This paper reviews the literature on the expression and activity of glucose transporter proteins-GLUT-1, GLUT-3, GLUT-4, GLUT-8, GLUT-9 and GLUT-12-in the human placenta, with a special focus on diabetes-complicated pregnancy. The characteristics of transporters in conditions of maternal normoglycemia and modifications occurring in the diabetic placenta are summarized, and the factors responsible for the regulation of the expression of selected isoforms are described. Finally, the impact of alterations in the placental expression of the aforementioned members of the GLUT family on intrauterine fetal development in pregnancies complicated by diabetes mellitus is discussed. Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.

  4. ABC transporter Cdr1p harbors charged residues in the intracellular loop and nucleotide-binding domain critical for protein trafficking and drug resistance.

    Science.gov (United States)

    Shah, Abdul Haseeb; Banerjee, Atanu; Rawal, Manpreet Kaur; Saxena, Ajay Kumar; Mondal, Alok Kumar; Prasad, Rajendra

    2015-08-01

    The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. The Rules and Functions of Nucleocytoplasmic Shuttling Proteins.

    Science.gov (United States)

    Fu, Xuekun; Liang, Chao; Li, Fangfei; Wang, Luyao; Wu, Xiaoqiu; Lu, Aiping; Xiao, Guozhi; Zhang, Ge

    2018-05-12

    Biological macromolecules are the basis of life activities. There is a separation of spatial dimension between DNA replication and RNA biogenesis, and protein synthesis, which is an interesting phenomenon. The former occurs in the cell nucleus, while the latter in the cytoplasm. The separation requires protein to transport across the nuclear envelope to realize a variety of biological functions. Nucleocytoplasmic transport of protein including import to the nucleus and export to the cytoplasm is a complicated process that requires involvement and interaction of many proteins. In recent years, many studies have found that proteins constantly shuttle between the cytoplasm and the nucleus. These shuttling proteins play a crucial role as transport carriers and signal transduction regulators within cells. In this review, we describe the mechanism of nucleocytoplasmic transport of shuttling proteins and summarize some important diseases related shuttling proteins.

  6. Intestinal Lymphangiectasia Secondary to Neuroblastoma

    Directory of Open Access Journals (Sweden)

    RM Reifen

    1994-01-01

    Full Text Available An eight month-old infant presented with a 10-day history of vomiting and diarrhea, and a one-week history of swelling of the lower extremities. Laboratory evaluations revealed hypoproteinemia and lymphocytopenia due to protein-losing enteropathy. Peroral small bowel biopsy showed intestinal lymphangiectasia. Subsequent onset of unexplained ecchymosis and obstructive jaundice resulted in additional studies which revealed an omental neuroblastoma as the underlying etiology of the infant’s symptoms. This report emphasizes the importance of considering secondary, obstructive causes for lymphangiectasia and protein-losing enteropathy.

  7. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inê s CR; Willige, Bjö rn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-01-01

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  8. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  9. Down-Regulation of the Na+-Coupled Phosphate Transporter NaPi-IIa by AMP-Activated Protein Kinase

    Directory of Open Access Journals (Sweden)

    Miribane Dërmaku-Sopjani

    2013-11-01

    Full Text Available Background/Aims: The Na+-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na+ gradient across the apical cell membrane, which is maintained by Na+ extrusion across the basolateral cell membrane through the Na+/K+ ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na+ accumulation and K+ loss with eventual decrease of cell membrane potential, Cl- entry and cell swelling. Upon energy depletion, early inhibition of Na+-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK, a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. Methods: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1-HA, of inactive AMPKαK45R (AMPKα1K45R+AMPKβ1-Flag+AMPKγ1-HA or of constitutively active AMPKγR70Q (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1R70Q. NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. Results: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM to the extracellular bath solution generated a current (Ip, which was significantly decreased by coexpression of wild-type AMPK and of AMPKγR70Q but not of AMPKαK45R. The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM. Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. Conclusion: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport.

  10. Plasma Homocysteine is Not Related to the Severity of Microangiopathy in Secondary Raynaud Phenomenon

    Science.gov (United States)

    Jacomella, Vincenzo; Wasila, Monika; Husmann, Marc; Gitzelmann, Gabriela; Meier, Thomas; Amann-Vesti, Beatrice

    2011-01-01

    Introduction: The role of elevated homocysteine in primary and secondary Raynaud phenomenon (RP) and in patients with atherosclerosis has been reported controversially. In secondary RP due to connective tissue disease specific alterations of nailfold capillaries might be present. An association between these microvascular changes and homocysteine has been suggested. Aim: The aim of this study was to determine whether homocysteine level differs between patients with primary and secondary RP and to test the hypothesis that homocysteine or other cardiovascular risk factors are associated with specific features of microangiopathy in secondary RP. Patients and Methods Eighty-one consecutive patients with RP referred for vascular assessment were studied by nailfold capillaroscopy. Homocysteine, C-reactive protein and cholesterol were measured and other cardiovascular risk factors and comorbidities assessed. Results: Homocysteine, C-reactive-protein and cholesterol levels did not differ between patients with primary (n=60) and secondary RP (n=21). Likewise, no differences in the prevalence of cardiovascular risk factors and comorbidities were found. In secondary RP no correlation was found between microvascular involvement and homocysteine or C-reactive protein. Conclusion: Plasma homocysteine is not different in patients with either primary or secondary RP and is therefore not a marker for the distinction of these diseases. The extent of microvascular involvement in secondary RP does not correlate with plasma homocysteine. PMID:22216066

  11. Intracellular loop 5 is important for the transport mechanism and molecular pharmacology of the human serotonin transporter

    DEFF Research Database (Denmark)

    Said, Saida; Neubauer, Henrik Amtoft; Müller, Heidi Kaastrup

    2015-01-01

    The serotonin transporter (SERT) belongs to a family of transport proteins called the neurotransmitter:sodium symporters. The specialized members of this family transport different neurotransmitters across the cell membrane, thereby regulating signaling between neurons. Most of these transporters...

  12. Enhanced Boron Tolerance in Plants Mediated by Bidirectional Transport Through Plasma Membrane Intrinsic Proteins.

    Science.gov (United States)

    Mosa, Kareem A; Kumar, Kundan; Chhikara, Sudesh; Musante, Craig; White, Jason C; Dhankher, Om Parkash

    2016-02-23

    High boron (B) concentration is toxic to plants that limit plant productivity. Recent studies have shown the involvement of the members of major intrinsic protein (MIP) family in controlling B transport. Here, we have provided experimental evidences showing the bidirectional transport activity of rice OsPIP1;3 and OsPIP2;6. Boron transport ability of OsPIP1;3 and OsPIP2;6 were displayed in yeast HD9 mutant strain (∆fps1∆acr3∆ycf1) as a result of increased B sensitivity, influx and accumulation by OsPIP1;3, and rapid efflux activity by OsPIP2;6. RT-PCR analysis showed strong upregulation of OsPIP1;3 and OsPIP2;6 transcripts in roots by B toxicity. Transgenic Arabidopsis lines overexpressing OsPIP1;3 and OsPIP2;6 exhibited enhanced tolerance to B toxicity. Furthermore, B concentration was significantly increased after 2 and 3 hours of tracer boron ((10)B) treatment. Interestingly, a rapid efflux of (10)B from the roots of the transgenic plants was observed within 1 h of (10)B treatment. Boron tolerance in OsPIP1;3 and OsPIP2;6 lines was inhibited by aquaporin inhibitors, silver nitrate and sodium azide. Our data proved that OsPIP1;3 and OsPIP2;6 are indeed involved in both influx and efflux of boron transport. Manipulation of these PIPs could be highly useful in improving B tolerance in crops grown in high B containing soils.

  13. Biomimetic materials for protein storage and transport

    Science.gov (United States)

    Firestone, Millicent A [Elmhurst, IL; Laible, Philip D [Villa Park, IL

    2012-05-01

    The invention provides a method for the insertion of protein in storage vehicles and the recovery of the proteins from the vehicles, the method comprising supplying isolated protein; mixing the isolated protein with a fluid so as to form a mixture, the fluid comprising saturated phospholipids, lipopolymers, and a surfactant; cycling the mixture between a first temperature and a second temperature; maintaining the mixture as a solid for an indefinite period of time; diluting the mixture in detergent buffer so as to disrupt the composition of the mixture, and diluting to disrupt the fluid in its low viscosity state for removal of the guest molecules by, for example, dialysis, filtering or chromatography dialyzing/filtering the emulsified solid.

  14. Humic Acid Confers HIGH-AFFINITY K+ TRANSPORTER 1-Mediated Salinity Stress Tolerance in Arabidopsis.

    Science.gov (United States)

    Khaleda, Laila; Park, Hee Jin; Yun, Dae-Jin; Jeon, Jong-Rok; Kim, Min Gab; Cha, Joon-Yung; Kim, Woe-Yeon

    2017-12-31

    Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on Arabidopsis thaliana seedlings. To identify the molecular mechanisms of HA-induced salt stress tolerance in Arabidopsis, we examined possible roles of a sodium influx transporter HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1). Salt-induced root growth inhibition in HKT1 overexpressor transgenic plants (HKT1-OX) was rescued by application of HA, but not in wild-type and other plants. Moreover, salt-induced degradation of HKT1 protein was blocked by HA treatment. In addition, the application of HA to HKT1-OX seedlings led to increased distribution of Na+ in roots up to the elongation zone and caused the reabsorption of Na+ by xylem and parenchyma cells. Both the influx of the secondary messenger calcium and its cytosolic release appear to function in the destabilization of HKT1 protein under salt stress. Taken together, these results suggest that HA could be applied to the field to enhance plant growth and salt stress tolerance via post-transcriptional control of the HKT1 transporter gene under saline conditions.

  15. Evidence of a gustatory-vestibular pathway for protein transport.

    Science.gov (United States)

    Gacek, Richard; Lyon, Michael J

    2010-02-01

    To demonstrate anatomically a pathway for protein transport from the palate to the vestibular system. The vestibulofacial anastomosis and associated ganglion cells were identified in a collection of 160 horizontally sectioned human temporal bones that had been stained with hematoxylin and eosin. Wheat germ agglutinin-horseradish peroxidase (HRP) was applied to the greater superficial petrosal nerve in 4 Sprague-Dawley rats. After 30 hours, the rats were killed by intracardiac perfusion, and the seventh and eighth nerves with adjacent brainstem removed. Frozen sections cut at 30 mum through this block were then reacted for HRP, counterstained with neutral red, and mounted on slides for examination in the light microscope. Thirty-two of the 160 human temporal bones contained sections through the vestibulofacial anastomosis and its ganglion. In all cases, the ganglion was incorporated into the vestibular ganglion (VG) adjacent to the nervus intermedius. In all 4 experimental rats, HRP reaction product labeled a small number of ganglion cells in the VG adjacent to the nervus intermedius and facial nerve. These observations support the presence of a pathway from receptors in the palate to the VG.

  16. LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.

    Science.gov (United States)

    Pelkonen, Laura; Sato, Kazuki; Reinisalo, Mika; Kidron, Heidi; Tachikawa, Masanori; Watanabe, Michitoshi; Uchida, Yasuo; Urtti, Arto; Terasaki, Tetsuya

    2017-03-06

    The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na + /K + ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.

  17. Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide.

    OpenAIRE

    Thomas, A P; Halestrap, A P

    1981-01-01

    1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis...

  18. Intracellular transport of fat-soluble vitamins A and E.

    Science.gov (United States)

    Kono, Nozomu; Arai, Hiroyuki

    2015-01-01

    Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E. © 2014 The Authors. Traffic published by John Wiley & Sons Ltd.

  19. Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar auxin transport.

    Science.gov (United States)

    Naramoto, Satoshi

    2017-12-01

    Directional cell-to-cell transport of functional molecules, called polar transport, enables plants to sense and respond to developmental and environmental signals. Transporters that localize to plasma membranes (PMs) in a polar manner are key components of these systems. PIN-FORMED (PIN) auxin efflux carriers, which are the most studied polar-localized PM proteins, are implicated in the polar transport of auxin that in turn regulates plant development and tropic growth. In this review, the regulatory mechanisms underlying polar localization of PINs, control of auxin efflux activity, and PIN abundance at PMs are considered. Up to date information on polar-localized nutrient transporters that regulate directional nutrient movement from soil into the root vasculature is also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Low-Protein Diet Supplemented with Keto Acids Is Associated with Suppression of Small-Solute Peritoneal Transport Rate in Peritoneal Dialysis Patients

    OpenAIRE

    Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Zhang, Weiming; Cao, Liou; Wang, Qin; Ni, Zhaohui; Yao, Qiang

    2011-01-01

    Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD) patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6–0.8 g/kg/d), keto-acid-supplemented low- (sLP: 0.6–0.8 g/kg/d with 0.12 g/kg/d of keto acids), or high- (HP: 1.0–1.2 g/kg/d) protein diet and lasted for one year. In this study, the variat...

  1. Imaging transport phenomena during lysozyme protein crystal growth by the hanging drop technique

    Science.gov (United States)

    Sethia Gupta, Anamika; Gupta, Rajive; Panigrahi, P. K.; Muralidhar, K.

    2013-06-01

    The present study reports the transport process that occurs during the growth of lysozyme protein crystals by the hanging drop technique. A rainbow schlieren technique has been employed for imaging changes in salt concentration. A one dimensional color filter is used to record the deflection of the light beam. An optical microscope and an X-ray crystallography unit are used to characterize the size, tetragonal shape and Bravais lattice constants of the grown crystals. A parametric study on the effect of drop composition, drop size, reservoir height and number of drops on the crystal size and quality is reported. Changes in refractive index are not large enough to create a meaningful schlieren image in the air gap between the drop and the reservoir. However, condensation of fresh water over the reservoir solution creates large changes in the concentration of NaCl, giving rise to clear color patterns in the schlieren images. These have been analyzed to obtain salt concentration profiles near the free surface of the reservoir solution as a function of time. The diffusion of fresh water into the reservoir solution at the early stages of crystal growth followed by the mass flux of salt from the bulk solution towards the free surface has been recorded. The overall crystal growth process can be classified into two regimes, as demarcated by the changes in slope of salt concentration within the reservoir. The salt concentration in the reservoir equilibrates at long times when the crystallization process is complete. Thus, transport processes in the reservoir emerge as the route to monitor protein crystal growth in the hanging drop configuration. Results show that crystal growth rate is faster for a higher lysozyme concentration, smaller drops, and larger reservoir heights.

  2. Gestational Protein Restriction Impairs Insulin-Regulated Glucose Transport Mechanisms in Gastrocnemius Muscles of Adult Male Offspring

    Science.gov (United States)

    Blesson, Chellakkan S.; Sathishkumar, Kunju; Chinnathambi, Vijayakumar

    2014-01-01

    Type II diabetes originates from various genetic and environmental factors. Recent studies showed that an adverse uterine environment such as that caused by a gestational low-protein (LP) diet can cause insulin resistance in adult offspring. The mechanism of insulin resistance induced by gestational protein restriction is not clearly understood. Our aim was to investigate the role of insulin signaling molecules in gastrocnemius muscles of gestational LP diet–exposed male offspring to understand their role in LP-induced insulin resistance. Pregnant Wistar rats were fed a control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery and a normal diet after weaning. Only male offspring were used in this study. Glucose and insulin responses were assessed after a glucose tolerance test. mRNA and protein levels of molecules involved in insulin signaling were assessed at 4 months in gastrocnemius muscles. Muscles were incubated ex vivo with insulin to evaluate insulin-induced phosphorylation of insulin receptor (IR), Insulin receptor substrate-1, Akt, and AS160. LP diet-fed rats gained less weight than controls during pregnancy. Male pups from LP diet–fed mothers were smaller but exhibited catch-up growth. Plasma glucose and insulin levels were elevated in LP offspring when subjected to a glucose tolerance test; however, fasting levels were comparable. LP offspring showed increased expression of IR and AS160 in gastrocnemius muscles. Ex vivo treatment of muscles with insulin showed increased phosphorylation of IR (Tyr972) in controls, but LP rats showed higher basal phosphorylation. Phosphorylation of Insulin receptor substrate-1 (Tyr608, Tyr895, Ser307, and Ser318) and AS160 (Thr642) were defective in LP offspring. Further, glucose transporter type 4 translocation in LP offspring was also impaired. A gestational LP diet leads to insulin resistance in adult offspring by a mechanism involving inefficient insulin-induced IR, Insulin receptor

  3. Cholestasis and regulation of genes related to drug metabolism and biliary transport in rat liver following treatment with cyclosporine A and sirolimus (Rapamycin)

    DEFF Research Database (Denmark)

    Bramow, S; Ott, P; Thomsen Nielsen, F

    2001-01-01

    then analysed as were hepatic mRNA levels of canalicular transport proteins (Mrp2, Bsep, Mdr1b and Mdr2), sinusoidal transport proteins (Ntcp, Oatp1 and Oatp2), GSH related enzymes (gamma-glutamylcysteine synthetase light (GCSlc) and heavy (GCShc) chain subunits and glutathione-S-transferase) and CYPs (CYP3A9...... secondary to cyclosporine A could be related to reduction in mRNA expression of GSH synthesising enzymes and Mrp2, leading to reduced protection against oxidative stress and reduced bile acid-independent bile flow. After sirolimus treatment, Mrp2 mRNA was also reduced together with reduced levels of most...... CYPs and increased Oatp2, possibly leading to accumulation of toxic metabolites in the hepatocytes. The enhanced cholestatic effect of the combination treatment could be related to reduced GSH synthesising enzymes and even more pronounced reduction in Mrp2 mRNA and increase of Oatp2 mRNA....

  4. Membrane permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release

    DEFF Research Database (Denmark)

    Rickhag, Karl Mattias; Owens, WA; Winkler, Marie-Therese

    2013-01-01

    The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate......-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects....

  5. Role of NH3 and NH4+ transporters in renal acid-base transport.

    Science.gov (United States)

    Weiner, I David; Verlander, Jill W

    2011-01-01

    Renal ammonia excretion is the predominant component of renal net acid excretion. The majority of ammonia excretion is produced in the kidney and then undergoes regulated transport in a number of renal epithelial segments. Recent findings have substantially altered our understanding of renal ammonia transport. In particular, the classic model of passive, diffusive NH3 movement coupled with NH4+ "trapping" is being replaced by a model in which specific proteins mediate regulated transport of NH3 and NH4+ across plasma membranes. In the proximal tubule, the apical Na+/H+ exchanger, NHE-3, is a major mechanism of preferential NH4+ secretion. In the thick ascending limb of Henle's loop, the apical Na+-K+-2Cl- cotransporter, NKCC2, is a major contributor to ammonia reabsorption and the basolateral Na+/H+ exchanger, NHE-4, appears to be important for basolateral NH4+ exit. The collecting duct is a major site for renal ammonia secretion, involving parallel H+ secretion and NH3 secretion. The Rhesus glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane, and its role in renal acid-base homeostasis is controversial. In the inner medullary collecting duct (IMCD), basolateral Na+-K+-ATPase enables active basolateral NH4+ uptake. In addition to these proteins, several other proteins also contribute to renal NH3/NH4+ transport. The role and mechanisms of these proteins are discussed in depth in this review.

  6. Effects of dietary nitrogen concentration on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in ruminal papillae from lactating Holstein cows

    DEFF Research Database (Denmark)

    Røjen, Betina Amdisen; Poulsen, Søren Brandt; Theil, Peter Kappel

    2011-01-01

    To test the hypothesis that dietary N concentrations affect gut epithelial urea transport by modifying the expression of urea transporter B (UT-B) and aquaporins (AQP), the mRNA expression and protein abundance of UT-B and AQP3, AQP7, AQP8, and AQP10 were investigated in ruminal papillae from 9...... lactating dairy cows. Ruminal papillae were harvested from cows fed low N (12.9% crude protein) and high N (17.1% crude protein) diets in a crossover design with 21-d periods. The mRNA expression was determined by real-time reverse transcription-PCR and protein abundance by immunoblotting. The m......RNA expression of UT-B was not affected by dietary treatment, whereas mRNA expression of AQP3, 7, and 10 were greater in the high N compared with the low N fed cows. Using peptide-derived rabbit antibodies to cow AQP3, 7, and 8, immunoblotting revealed bands of approximately 27, 27, and 24 kDa in ruminal...

  7. Graphene oxide as a protein matrix: influence on protein biophysical properties.

    Science.gov (United States)

    Hernández-Cancel, Griselle; Suazo-Dávila, Dámaris; Ojeda-Cruzado, Axel J; García-Torres, Desiree; Cabrera, Carlos R; Griebenow, Kai

    2015-10-19

    This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

  8. On the analysis of protein-protein interactions via knowledge-based potentials for the prediction of protein-protein docking

    DEFF Research Database (Denmark)

    Feliu, Elisenda; Aloy, Patrick; Oliva, Baldo

    2011-01-01

    Development of effective methods to screen binary interactions obtained by rigid-body protein-protein docking is key for structure prediction of complexes and for elucidating physicochemical principles of protein-protein binding. We have derived empirical knowledge-based potential functions for s...... and with independence of the partner. This information is encoded at the residue level and could be easily incorporated in the initial grid scoring for Fast Fourier Transform rigid-body docking methods.......Development of effective methods to screen binary interactions obtained by rigid-body protein-protein docking is key for structure prediction of complexes and for elucidating physicochemical principles of protein-protein binding. We have derived empirical knowledge-based potential functions...... for selecting rigid-body docking poses. These potentials include the energetic component that provides the residues with a particular secondary structure and surface accessibility. These scoring functions have been tested on a state-of-art benchmark dataset and on a decoy dataset of permanent interactions. Our...

  9. A Program for Iron Economy during Deficiency Targets Specific Fe Proteins.

    Science.gov (United States)

    Hantzis, Laura J; Kroh, Gretchen E; Jahn, Courtney E; Cantrell, Michael; Peers, Graham; Pilon, Marinus; Ravet, Karl

    2018-01-01

    Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis ( Arabidopsis thaliana ) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome- b 6 f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency. © 2018 American Society of Plant Biologists. All Rights Reserved.

  10. Flavonoids act as negative regulators of auxin transport in vivo in arabidopsis

    Science.gov (United States)

    Brown, D. E.; Rashotte, A. M.; Murphy, A. S.; Normanly, J.; Tague, B. W.; Peer, W. A.; Taiz, L.; Muday, G. K.

    2001-01-01

    Polar transport of the plant hormone auxin controls many aspects of plant growth and development. A number of synthetic compounds have been shown to block the process of auxin transport by inhibition of the auxin efflux carrier complex. These synthetic auxin transport inhibitors may act by mimicking endogenous molecules. Flavonoids, a class of secondary plant metabolic compounds, have been suggested to be auxin transport inhibitors based on their in vitro activity. The hypothesis that flavonoids regulate auxin transport in vivo was tested in Arabidopsis by comparing wild-type (WT) and transparent testa (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis, chalcone synthase. In a comparison between tt4 and WT plants, phenotypic differences were observed, including three times as many secondary inflorescence stems, reduced plant height, decreased stem diameter, and increased secondary root development. Growth of WT Arabidopsis plants on naringenin, a biosynthetic precursor to those flavonoids with auxin transport inhibitor activity in vitro, leads to a reduction in root growth and gravitropism, similar to the effects of synthetic auxin transport inhibitors. Analyses of auxin transport in the inflorescence and hypocotyl of independent tt4 alleles indicate that auxin transport is elevated in plants with a tt4 mutation. In hypocotyls of tt4, this elevated transport is reversed when flavonoids are synthesized by growth of plants on the flavonoid precursor, naringenin. These results are consistent with a role for flavonoids as endogenous regulators of auxin transport.

  11. Functional structural motifs for protein-ligand, protein-protein, and protein-nucleic acid interactions and their connection to supersecondary structures.

    Science.gov (United States)

    Kinjo, Akira R; Nakamura, Haruki

    2013-01-01

    Protein functions are mediated by interactions between proteins and other molecules. One useful approach to analyze protein functions is to compare and classify the structures of interaction interfaces of proteins. Here, we describe the procedures for compiling a database of interface structures and efficiently comparing the interface structures. To do so requires a good understanding of the data structures of the Protein Data Bank (PDB). Therefore, we also provide a detailed account of the PDB exchange dictionary necessary for extracting data that are relevant for analyzing interaction interfaces and secondary structures. We identify recurring structural motifs by classifying similar interface structures, and we define a coarse-grained representation of supersecondary structures (SSS) which represents a sequence of two or three secondary structure elements including their relative orientations as a string of four to seven letters. By examining the correspondence between structural motifs and SSS strings, we show that no SSS string has particularly high propensity to be found interaction interfaces in general, indicating any SSS can be used as a binding interface. When individual structural motifs are examined, there are some SSS strings that have high propensity for particular groups of structural motifs. In addition, it is shown that while the SSS strings found in particular structural motifs for nonpolymer and protein interfaces are as abundant as in other structural motifs that belong to the same subunit, structural motifs for nucleic acid interfaces exhibit somewhat stronger preference for SSS strings. In regard to protein folds, many motif-specific SSS strings were found across many folds, suggesting that SSS may be a useful description to investigate the universality of ligand binding modes.

  12. Functional discrimination of membrane proteins using machine learning techniques

    Directory of Open Access Journals (Sweden)

    Yabuki Yukimitsu

    2008-03-01

    Full Text Available Abstract Background Discriminating membrane proteins based on their functions is an important task in genome annotation. In this work, we have analyzed the characteristic features of amino acid residues in membrane proteins that perform major functions, such as channels/pores, electrochemical potential-driven transporters and primary active transporters. Results We observed that the residues Asp, Asn and Tyr are dominant in channels/pores whereas the composition of hydrophobic residues, Phe, Gly, Ile, Leu and Val is high in electrochemical potential-driven transporters. The composition of all the amino acids in primary active transporters lies in between other two classes of proteins. We have utilized different machine learning algorithms, such as, Bayes rule, Logistic function, Neural network, Support vector machine, Decision tree etc. for discriminating these classes of proteins. We observed that most of the algorithms have discriminated them with similar accuracy. The neural network method discriminated the channels/pores, electrochemical potential-driven transporters and active transporters with the 5-fold cross validation accuracy of 64% in a data set of 1718 membrane proteins. The application of amino acid occurrence improved the overall accuracy to 68%. In addition, we have discriminated transporters from other α-helical and β-barrel membrane proteins with the accuracy of 85% using k-nearest neighbor method. The classification of transporters and all other proteins (globular and membrane showed the accuracy of 82%. Conclusion The performance of discrimination with amino acid occurrence is better than that with amino acid composition. We suggest that this method could be effectively used to discriminate transporters from all other globular and membrane proteins, and classify them into channels/pores, electrochemical and active transporters.

  13. 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.

  14. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  15. The Inclusion of Chitosan in Poly-ε-caprolactone Nanoparticles: Impact on the Delivery System Characteristics and on the Adsorbed Ovalbumin Secondary Structure.

    Science.gov (United States)

    Jesus, Sandra; Fragal, Elizangela H; Rubira, Adley F; Muniz, Edvani C; Valente, Artur J M; Borges, Olga

    2018-01-01

    This report extensively explores the benefits of including chitosan into poly-ε-caprolactone (PCL) nanoparticles (NPs) to obtain an improved protein/antigen delivery system. Blend NPs (PCL/chitosan NPs) showed improved protein adsorption efficacy (84%) in low shear stress and aqueous environment, suggesting that a synergistic effect between PCL hydrophobic nature and the positive charges of chitosan present at the particle surface was responsible for protein interaction. Additionally, thermal analysis suggested the blend NPs were more stable than the isolated polymers and cytotoxicity assays in a primary cell culture revealed chitosan inclusion in PCL NPs reduced the toxicity of the delivery system. A quantitative 6-month stability study showed that the inclusion of chitosan in PCL NPs did not induce a change in adsorbed ovalbumin (OVA) secondary structure characterized by the increase in the unordered conformation (random coil), as it was observed for OVA adsorbed to chitosan NPs. Additionally, the slight conformational changes occurred, are not expected to compromise ovalbumin secondary structure and activity, during a 6-month storage even at high temperatures (45°C). In simulated biological fluids, PCL/chitosan NPs showed an advantageous release profile for oral delivery. Overall, the combination of PCL and chitosan characteristics provide PCL/chitosan NPs valuable features particularly important to the development of vaccines for developing countries, where it is difficult to ensure cold chain transportation and non-parenteral formulations would be preferred.

  16. JABAWS 2.2 distributed web services for Bioinformatics: protein disorder, conservation and RNA secondary structure.

    Science.gov (United States)

    Troshin, Peter V; Procter, James B; Sherstnev, Alexander; Barton, Daniel L; Madeira, Fábio; Barton, Geoffrey J

    2018-06-01

    JABAWS 2.2 is a computational framework that simplifies the deployment of web services for Bioinformatics. In addition to the five multiple sequence alignment (MSA) algorithms in JABAWS 1.0, JABAWS 2.2 includes three additional MSA programs (Clustal Omega, MSAprobs, GLprobs), four protein disorder prediction methods (DisEMBL, IUPred, Ronn, GlobPlot), 18 measures of protein conservation as implemented in AACon, and RNA secondary structure prediction by the RNAalifold program. JABAWS 2.2 can be deployed on a variety of in-house or hosted systems. JABAWS 2.2 web services may be accessed from the Jalview multiple sequence analysis workbench (Version 2.8 and later), as well as directly via the JABAWS command line interface (CLI) client. JABAWS 2.2 can be deployed on a local virtual server as a Virtual Appliance (VA) or simply as a Web Application Archive (WAR) for private use. Improvements in JABAWS 2.2 also include simplified installation and a range of utility tools for usage statistics collection, and web services querying and monitoring. The JABAWS CLI client has been updated to support all the new services and allow integration of JABAWS 2.2 services into conventional scripts. A public JABAWS 2 server has been in production since December 2011 and served over 800 000 analyses for users worldwide. JABAWS 2.2 is made freely available under the Apache 2 license and can be obtained from: http://www.compbio.dundee.ac.uk/jabaws. g.j.barton@dundee.ac.uk.

  17. Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells.

    Science.gov (United States)

    Matsui, T; Nakayama, H; Yoshida, K; Shinmyo, A

    2003-10-01

    Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.

  18. Natural polyphenols: Influence on membrane transporters

    Directory of Open Access Journals (Sweden)

    Saad Abdulrahman Hussain

    2016-03-01

    Full Text Available Accumulated evidences have focused on the use of natural polyphenolic compounds as nutraceuticals, since they showed a wide range of bioactivities and exhibited protection against variety of age related disorders. Polyphenols have variable potencies to interact, and hence alter the activities of various transporter proteins, many of them classified as ATP-Binding Cassette transporters, like multidrug resistance protein (MDRP, and p-glycoprotein (P-gp. Some of the efflux transporters are generally linked with anticancer and antiviral drug resistance; in this context, polyphenols may be beneficial in modulating drug resistance by increasing the efficacy of anticancer and antiviral drugs. Additionally, these effects were implicated to explain the influence of dietary polyphenols on drug efficacy as result of food-drug interactions. However, limited data are available about the influence of these components on uptake transporters. Therefore, the objective of this article is to review the potential efficacies of polyphenols in modulating the functional integrity of uptake transporter proteins, including those terminated the effect of neurotransmitters, and their possible influence in neuropharmacology. [J Complement Med Res 2016; 5(1.000: 97-104

  19. PWR.2 - the unique transportation

    International Nuclear Information System (INIS)

    Howell, G.E.; Wills, L.I.

    1987-01-01

    Design studies of the prototype machinery and installation of same to be used for test and evaluation of a new design of nuclear power plant for submarines, showed that there were advantages if large units could be fitted out entirely at the manufacturer's base in Barrow-in-Furness. However, they had then to be shipped to the customer at the Vulcan Naval Reactor Test Establishment in Caithness, Scotland. The transportation of the loads involved is described. The main loads were the primary unit which weighed 1300 tonnes and the secondary unit which was transported as five separate assemblies, the largest two of which weighed 151 and 43 tonnes. Five basic transportation methods were used: skidding on PTFE pads, sea passage with barge on submersible barge, rolling on airbags, skating on water skates and lifting and rolling on multi-wheeled trailers. By careful planning the primary unit was moved in 16 days and the secondary unit in 19 days. The route and methods used are described and illustrated. (U.K.)

  20. Enhancement in secondary particulate matter production due to mountain trapping

    Science.gov (United States)

    Yao, Teng; Fung, J. C. H.; Ma, H.; Lau, A. K. H.; Chan, P. W.; Yu, J. Z.; Xue, J.

    2014-10-01

    As China's largest economic development zone, the Pearl River Delta (PRD) is subject to particulate matter (PM) and visibility deterioration problems. Due to high PM concentration, haze days impacting ambient visibility have occurred frequently in this region. Besides visibility impairment, PM pollution also causes a negative impact on public health. These negative impacts have heightened the need to improve our understanding of the PM pollution of the PRD region. One major cause of the PRD pollution problem is cold front passages in the winter; however, the mechanism of pollution formation stays unclear. In this study, the Comprehensive Air Quality Model (CAMx) is utilized to investigate the detailed PM production and transport mechanisms in the PRD. Simulated concentrations of PM2.5 species, which have a good correlation with observation, show that sulfate and nitrate are the dominant pollutants among different PM2.5 species. Before the cold front passage a large amount of gas-phase and particle-phase pollutants are transported to the mountainous regions in the north of the PRD, and become trapped by the terrain. Over the mountain regions, cloud driven by upwelling flow promotes aqueous-phase reactions including oxidations of PM precursors such as SO2 and NO2. By this process, production of secondary PM is enhanced. When the cold front continues to advance further south, PM is transported to the PRD cities, and suppressed into a thin layer near the ground by a low planetary boundary layer (PBL). Thus high PM concentration episodes take place in the PRD cities. After examining production and transportation pathways, this study presents that the complex terrain configuration would block pollutant dispersion, provide cloudy environment, and advance secondary PM production. Previous studies have pointed out that pollution emitted from outside this region largely influences the air quality in the PRD; however, this study shows that pollutants from the outside could be

  1. ABC transporters in fish species: a review

    Directory of Open Access Journals (Sweden)

    Marta eFerreira

    2014-07-01

    Full Text Available ATP-binding cassette (ABC proteins were first recognized for their role in multidrug resistance (MDR in chemotherapeutic treatments, which is a major impediment for the successful treatment of many forms of malignant tumors in humans. These proteins, highly conserved throughout vertebrate species, were later related to cellular detoxification and accounted as responsible for protecting aquatic organisms from xenobiotic insults in the so-called multixenobiotic resistance mechanism (MXR. In recent years, research on these proteins in aquatic species has highlighted their importance in the detoxification mechanisms in fish thus it is of extreme added value to continue these studies. Several transporters have been pointed out as relevant in the ecotoxicological context associated to the transport of xenobiotics, such as P-glycoproteins (Pgps, multidrug-resistance-associated proteins (MRPs 1-5 and breast resistance associated protein (BCRP. In mammals, several nuclear receptors have been identified as mediators of phase I and II metabolizing enzymes and ABC transporters. In aquatic species, knowledge on co-regulation of detoxification mechanism is scarce and needs to be addressed. The interaction of emergent contaminants, with chemosensitizer potential, with ABC transporters in aquatic organisms can compromise detoxification processes and have population effects and should be studied in more detail. This review intends to summarize the recent advances in research on MXR mechanisms in fish species, focusing in 1 regulation and functioning of ABC proteins; 2 cooperation with phase I and II biotransformation enzymes; and 3 ecotoxicological relevance and information on emergent pollutants with ability to modulate ABC transporters expression and activity. Several lines of evidence are clear suggesting the important role of these transporters in detoxification mechanisms and must be further investigated in fish.

  2. Molecular Dynamics Investigation of Cl− and Water Transport through a Eukaryotic CLC Transporter

    OpenAIRE

    Cheng, Mary Hongying; Coalson, Rob D.

    2012-01-01

    Early crystal structures of prokaryotic CLC proteins identified three Cl– binding sites: internal (Sint), central (Scen), and external (Sext). A conserved external GLU (GLUex) residue acts as a gate competing for Sext. Recently, the first crystal structure of a eukaryotic transporter, CmCLC, revealed that in this transporter GLUex competes instead for Scen. Here, we use molecular dynamics simulations to investigate Cl– transport through CmCLC. The gating and Cl–/H+ transport cycle are inferre...

  3. Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system

    Directory of Open Access Journals (Sweden)

    Lalonde Sylvie

    2003-03-01

    Full Text Available Abstract Background The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact. Results Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters. Conclusions The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.

  4. Role of the Fur regulon in iron transport in Bacillus subtilis.

    Science.gov (United States)

    Ollinger, Juliane; Song, Kyung-Bok; Antelmann, Haike; Hecker, Michael; Helmann, John D

    2006-05-01

    The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding approximately 40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT.

  5. Amino Acid Molecular Units: Building Primary and Secondary Protein Structures

    Directory of Open Access Journals (Sweden)

    Aparecido R. Silva

    2008-05-01

    Full Text Available In order to guarantee the learning quality and suitable knowledge  use  about structural biology, it is fundamental to  exist, since the beginning of  students’ formation, the possibility of clear visualization of biomolecule structures. Nevertheless, the didactic books can only bring  schematic  drawings; even more elaborated figures and graphic computation  do not permit the necessary interaction.  The representation of three-dimensional molecular structures with ludic models, built with representative units, have supplied to the students and teachers a successfully experience to  visualize such structures and correlate them to the real molecules.  The design and applicability of the representative units were discussed with researchers and teachers before mould implementation.  In this stage  it  will be presented the  developed  kit  containing the  representative  plastic parts of the main amino acids.  The kit can demonstrate the interaction among the amino acids  functional groups  (represented by colors, shapes,  sizes and  the peptidic bonds between them  facilitating the assembly and visuali zation of the primary and secondary protein structure.  The models were designed for  Ca,  amino,  carboxyl groups  and  hydrogen. The  lateral chains have  well defined models that represent their geometrical shape.  The completed kit set  will be presented in this meeting (patent requested.  In the last phase of the project will be realized  an effective evaluation  of the kit  as a facilitative didactic tool of the teaching/learning process in the Structural Molecular Biology area.

  6. FTIR study of secondary structure of bovine serum albumin and ovalbumin

    International Nuclear Information System (INIS)

    Abrosimova, K V; Shulenina, O V; Paston, S V

    2016-01-01

    Proteins structure is the critical factor for their functioning. Fourier transform infrared spectroscopy provides a possibility to obtain information about secondary structure of proteins in different states and also in a whole biological samples. Infrared spectra of egg white from the untreated and hard-boiled hen's egg, and also of chicken ovalbumin and bovine serum albumin in lyophilic form and in aqueous solution were studied. Lyophilization of investigated globular proteins is accompanied by the decrease of a-helix structures and the increase in amount of intermolecular β-sheets. Analysis of infrared spectrum of egg white allowed to make an estimation of OVA secondary structure and to observe α-to-β structural transformation as a result of the heat denaturation. (paper)

  7. N-linked glycans do not affect plasma membrane localization of multidrug resistance protein 4 (MRP4) but selectively alter its prostaglandin E2 transport activity.

    Science.gov (United States)

    Miah, M Fahad; Conseil, Gwenaëlle; Cole, Susan P C

    2016-01-22

    Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Uncertainty analysis for secondary energy distributions

    International Nuclear Information System (INIS)

    Gerstl, S.A.W.

    1978-01-01

    In many transport calculations the integral design parameter of interest (response) is determined mainly by secondary particles such as gamma rays from (n,γ) reactions or secondary neutrons from inelastic scattering events or (n,2n) reactions. Standard sensitivity analysis usually allows to calculate the sensitivities to the production cross sections of such secondaries, but an extended formalism is needed to also obtain the sensitivities to the energy distribution of the generated secondary particles. For a 30-group standard cross-section set 84% of all non-zero table positions pertain to the description of secondary energy distributions (SED's) and only 16% to the actual reaction cross sections. Therefore, any sensitivity/uncertainty analysis which does not consider the effects of SED's is incomplete and neglects most of the input data. This paper describes the methods of how sensitivity profiles for SED's are obtained and used to estimate the uncertainty of an integral response due to uncertainties in these SED's. The detailed theory is documented elsewhere and implemented in the LASL sensitivity code SENSIT. SED sensitivity profiles have proven particularly valuable in cross-section uncertainty analyses for fusion reactors. Even when the production cross sections for secondary neutrons were assumed to be without error, the uncertainties in the energy distribution of these secondaries produced appreciable uncertainties in the calculated tritium breeding rate. However, complete error files for SED's are presently nonexistent. Therefore, methods will be described that allow rough error estimates due to estimated SED uncertainties based on integral SED sensitivities

  9. ATP-binding cassette (ABC) transporters in normal and pathological lung

    NARCIS (Netherlands)

    van der Deen, M; de Vries, EGE; Timens, W; Scheper, RJ; Timmer-Bosscha, H; Postma, DS

    2005-01-01

    ATP-binding cassette ( ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein ( P-gp), multidrug resistance-associated protein 1 ( MRP1) and

  10. Protein Structure Prediction by Protein Threading

    Science.gov (United States)

    Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

    The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

  11. Comparative genomics of transport proteins in probiotic and pathogenic Escherichia coli and Salmonella enterica strains.

    Science.gov (United States)

    Do, Jimmy; Zafar, Hassan; Saier, Milton H

    2017-06-01

    Escherichia coli is a genetically diverse species that can be pathogenic, probiotic, commensal, or a harmless laboratory strain. Pathogenic strains of E. coli cause urinary tract infections, diarrhea, hemorrhagic colitis, and pyelonephritis, while the two known probiotic E. coli strains combat inflammatory bowel disease and play a role in immunomodulation. Salmonella enterica, a close relative of E. coli, includes two important pathogenic serovars, Typhi and Typhimurium, causing typhoid fever and enterocolitis in humans, respectively, with the latter strain also causing a lethal typhoid fever-like disease in mice. In this study, we identify the transport systems and their substrates within seven E. coli strains: two probiotic strains, two extracellular pathogens, two intracellular pathogens, and K-12, as well as the two intracellular pathogenic S. enterica strains noted above. Transport systems characteristic of each probiotic or pathogenic species were thus identified, and the tabulated results obtained with all of these strains were compared. We found that the probiotic and pathogenic strains generally contain more iron-siderophore and sugar transporters than E. coli K-12. Pathogens have increased numbers of pore-forming toxins, protein secretion systems, decarboxylation-driven Na + exporters, electron flow-driven monovalent cation exporters, and putative transporters of unknown function compared to the probiotic strains. Both pathogens and probiotic strains encode metabolite transporters that reflect their intracellular versus extracellular environments. The results indicate that the probiotic strains live extracellularly. It seems that relatively few virulence factors can convert a beneficial or commensal microorganism into a pathogen. Taken together, the results reveal the distinguishing features of these strains and provide a starting point for future engineering of beneficial enteric bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Softwood sawlog secondary transport travel speed prediction for the ...

    African Journals Online (AJOL)

    The objective of this study was to develop a predictive model for travel speed of softwood sawlog timber transport (STT) vehicles over a range of forest and provincial roads of varying condition for the South African forestry industry. Data was accumulated from both the Eastern Cape/KwaZulu-Natal and Mpumalanga forest ...

  13. Transport of Magnesium by a Bacterial Nramp-Related Gene

    Science.gov (United States)

    Rodionov, Dmitry A.; Freedman, Benjamin G.; Senger, Ryan S.; Winkler, Wade C.

    2014-01-01

    Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5–2.0 mM). Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes. PMID:24968120

  14. Placenta Copper Transport Proteins in Preeclampsia

    Science.gov (United States)

    Placental insufficiency underlying preeclampsia (PE) is associated with impaired placental angiogenesis. As copper (Cu) is essential to angiogenesis, we investigated differences in the expression of placental Cu transporters Menkes (ATP7A), Wilsons (ATP7B) and the Cu chaperone (CCS) for superoxide d...

  15. PROTEOTRONICS: The emerging science of protein-based electronic devices

    International Nuclear Information System (INIS)

    Alfinito, Eleonora; Pousset, Jeremy; Reggiani, Lino

    2015-01-01

    Protein-mediated charge transport is of relevant importance in the design of protein based electronics and in attaining an adequate level of understanding of protein functioning. This is particularly true for the case of transmembrane proteins, like those pertaining to the G protein coupled receptors (GPCRs). These proteins are involved in a broad range of biological processes like catalysis, substance transport, etc., thus being the target of a large number of clinically used drugs. This paper briefly reviews a variety of experiments devoted to investigate charge transport in proteins and present a unified theoretical model able to relate macroscopic experimental results with the conformations of the amino acids backbone of the single protein. (paper)

  16. The Endoplasmic Reticulum Coat Protein II Transport Machinery Coordinates Cellular Lipid Secretion and Cholesterol Biosynthesis*

    Science.gov (United States)

    Fryer, Lee G. D.; Jones, Bethan; Duncan, Emma J.; Hutchison, Claire E.; Ozkan, Tozen; Williams, Paul A.; Alder, Olivia; Nieuwdorp, Max; Townley, Anna K.; Mensenkamp, Arjen R.; Stephens, David J.; Dallinga-Thie, Geesje M.; Shoulders, Carol C.

    2014-01-01

    Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions. PMID:24338480

  17. Development of protein biomarkers in cerebrospinal fluid for secondary progressive multiple sclerosis using selected reaction monitoring mass spectrometry (SRM-MS

    Directory of Open Access Journals (Sweden)

    Jia Yan

    2012-07-01

    Full Text Available Abstract Background Multiple sclerosis (MS is a chronic inflammatory disorder of the central nervous system (CNS. It involves damage to the myelin sheath surrounding axons and to the axons themselves. MS most often presents with a series of relapses and remissions but then evolves over a variable period of time into a slowly progressive form of neurological dysfunction termed secondary progressive MS (SPMS. The reasons for this change in clinical presentation are unclear. The absence of a diagnostic marker means that there is a lag time of several years before the diagnosis of SPMS can be established. At the same time, understanding the mechanisms that underlie SPMS is critical to the development of rational therapies for this untreatable stage of the disease. Results Using high performance liquid chromatography-coupled mass spectrometry (HPLC; we have established a highly specific and sensitive selected reaction monitoring (SRM assay. Our multiplexed SRM assay has facilitated the simultaneous detection of surrogate peptides originating from 26 proteins present in cerebrospinal fluid (CSF. Protein levels in CSF were generally ~200-fold lower than that in human sera. A limit of detection (LOD was determined to be as low as one femtomol. We processed and analysed CSF samples from a total of 22 patients with SPMS, 7 patients with SPMS treated with lamotrigine, 12 patients with non-inflammatory neurological disorders (NIND and 10 healthy controls (HC for the levels of these 26 selected potential protein biomarkers. Our SRM data found one protein showing significant difference between SPMS and HC, three proteins differing between SPMS and NIND, two proteins between NIND and HC, and 11 protein biomarkers showing significant difference between a lamotrigine-treated and untreated SPMS group. Principal component analysis (PCA revealed that these 26 proteins were correlated, and could be represented by four principal components. Overall, we established an

  18. Participation of the oviductal s100 calcium binding protein G in the genomic effect of estradiol that accelerates oviductal embryo transport in mated rats

    Directory of Open Access Journals (Sweden)

    Croxatto Horacio B

    2011-05-01

    Full Text Available Abstract Background Mating changes the mechanism by which E2 regulates oviductal egg transport, from a non-genomic to a genomic mode. Previously, we found that E2 increased the expression of several genes in the oviduct of mated rats, but not in unmated rats. Among the transcripts that increased its level by E2 only in mated rats was the one coding for an s100 calcium binding protein G (s100 g whose functional role in the oviduct is unknown. Methods Herein, we investigated the participation of s100 g on the E2 genomic effect that accelerates oviductal transport in mated rats. Thus, we determined the effect of E2 on the mRNA and protein level of s100 g in the oviduct of mated and unmated rats. Then, we explored the effect of E2 on egg transport in unmated and mated rats under conditions in which s100 g protein was knockdown in the oviduct by a morpholino oligonucleotide against s100 g (s100 g-MO. In addition, the localization of s100 g in the oviduct of mated and unmated rats following treatment with E2 was also examined. Results Expression of s100 g mRNA progressively increased at 3-24 h after E2 treatment in the oviduct of mated rats while in unmated rats s100 g increased only at 12 and 24 hours. Oviductal s100 g protein increased 6 h following E2 and continued elevated at 12 and 24 h in mated rats, whereas in unmated rats s100 g protein increased at the same time points as its transcript. Administration of a morpholino oligonucleotide against s100 g transcript blocked the effect of E2 on egg transport in mated, but not in unmated rats. Finally, immunoreactivity of s100 g was observed only in epithelial cells of the oviducts of mated and unmated rats and it was unchanged after E2 treatment. Conclusions Mating affects the kinetic of E2-induced expression of s100 g although it not changed the cellular localization of s100 g in the oviduct after E2 . On the other hand, s100 g is a functional component of E2 genomic effect that accelerates egg

  19. A method of radiocompetitive assay of total thyroxine in the serum by means of enzymatic release of thyroxine from the transporting proteins

    International Nuclear Information System (INIS)

    Snarski, A.; Wyrwinski, J.

    1978-01-01

    Pepsin causes denaturation of the transporting proteins and liberates thyroxine which can be assayed by the radiocompetitive method. Change of the pH of the medium from acid to alkaline inactivates irreveribly pepsin. The enzymatic release of thyroxine is much simpler that the method of ethanol extraction and thermal denaturation of the transporting proteins applied up to now. The new technique of thyroxine release has been introduced for radiocompetitive determination of thyroxine using dextran coated charcoal for adsorption of the free hormone. A new method has been elaborated for preparation of working standards of thyroxine in a mixture of pepsin solution with hormone-free serum. The method is efficient and rapid. The normal range is from 50 to 130 nanomol/l. Over 7 000 determinations were done as yet in patients with suspected thyroid function disturbances. (author)

  20. Expression of heat shock protein 70 in transport-stressed broiler pectoralis major muscle and its relationship with meat quality.

    Science.gov (United States)

    Xing, T; Wang, M F; Han, M Y; Zhu, X S; Xu, X L; Zhou, G H

    2017-09-01

    Omics research has indicated that heat shock protein 70 (HSP70) is a potential biomarker of meat quality. However, the specific changes and the potential role of HSP70 in postmortem meat quality development need to be further defined. In this study, Arbor Acres broiler chickens (n=126) were randomly categorized into three treatment groups of unstressed control (C), 0.5-h transport (T) and subsequent water shower spray following transport (T/W). Each treatment consisted of six replicates with seven birds each. The birds were transported according to a designed protocol. The pectoralis major (PM) muscles of the transport-stressed broilers were categorized as normal and pale, soft and exudative (PSE)-like muscle samples according to L* and pH24 h values to test the expression and location of HSP70. Results revealed that the activities of plasma creatine kinase and lactate dehydrogenase increased significantly (Pmeat quality and stress indicators. In conclusion, this research suggests that the variation in HSP70 expression may provide a novel insight into the pathways underlying meat quality development.

  1. Effect of levofloxacin, pazufloxacin, enrofloxacin, and meloxicam on the immunolocalization of ABCG-2 transporter protein in rabbit retina.

    Science.gov (United States)

    Khan, Adil Mehraj; Rampal, Satyavan; Sood, Naresh Kumar

    2018-03-01

    Adenosine triphosphate-binding cassette (ABC) sub-family G member-2 (ABCG-2) is a transporter protein, implicated for multi-drug efflux from tissues. This study evaluated the effect of fluoroquinolones; levofloxacin, pazufloxacin and enrofloxacin, and non-steroidal anti-inflammatory drug, meloxicam; on the immunolocalization of ABCG-2 transporter protein of rabbit retinas. Thirty-two male rabbits were randomly divided in to eight groups. Control group was gavaged, 2% benzyl alcohol in 5% dextrose since these chemicals are excipients of the drug preparations used in the treatment groups of this study. Four groups were exclusively gavaged, levofloxacin hemihydrate (10 mg/kg body weight b.i.d 12 h), pazufloxacin mesylate (10 mg/kg body weight b.i.d 12 h), enrofloxacin (20 mg/kg body weight o.d.), and meloxicam (0.2 mg/kg body weight o.d.), respectively. Three other groups were co-gavaged meloxicam with above fluoroquinolones, respectively. These drugs were administered for 21 days. ABCG-2 immunolocalization was mild in the retinas of control and levofloxacin-alone-treated groups. The immunolocalization intensity was significantly higher in meloxicam-alone-treated group when compared to control and levofloxacin-alone-treated groups. Immunolocalization of this transporter increased in the levofloxacin-meloxicam co-treated group when compared to the levofloxacin-alone-treated group. Highest immunolocalization was observed in the enrofloxacin-meloxicam co-treated group although the immunolocalization of all treatment groups, except the levofloxacin-alone-treated group, was significantly higher than the control and levofloxacin-alone-treated groups.

  2. Versatile secondary beam for the meson area

    International Nuclear Information System (INIS)

    Kirk, T.

    1982-03-01

    A new secondary beam design is outlined for the Meson M6 Beamline that combines versatility with economy. The beamline described will transport charged particles of either sign to 800 GeV/c and bring the beam to a focus in one of three potential experimental areas. The plan makes maximal use of existing civil construction

  3. The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

    International Nuclear Information System (INIS)

    Lim, Hyoun-Sub; Nam, Jiryun; Seo, Eun-Young; Nam, Moon; Vaira, Anna Maria; Bae, Hanhong; Jang, Chan-Yong; Lee, Cheol Ho; Kim, Hong Gi; Roh, Mark; Hammond, John

    2014-01-01

    Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP SP ) with that from AltMV-Po (CP Po ) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP Po [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP SP but not CP Po interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP SP than CP Po in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. - Highlights: • Alternanthera mosaic virus CP is an elicitor of systemic necrosis in N. benthamiana. • Virus-induced systemic necrosis is enhanced at 15 °C compared to 25 °C. • Induction of systemic necrosis is dependent on as few as two CP amino acid residues. • These residues are at subunit interfaces within the same turn of the virion helix. • Inducer/non-inducer CPs interact differentially with a boron transporter protein

  4. Plant protein and secondary metabolites influence diet selection in a mammalian specialist herbivore

    Science.gov (United States)

    Ulappa, Amy C.; Kelsey, Rick G.; Frye, Graham G.; Rachlow, Janet L.; Shipley, Lisa A.; Bond, Laura; Pu, Xinzhu; Forbey, Jennifer Sorensen

    2015-01-01

    For herbivores, nutrient intake is limited by the relatively low nutritional quality of plants and high concentrations of potentially toxic defensive compounds (plant secondary metabolites, PSMs) produced by many plants. In response to phytochemical challenges, some herbivores selectively forage on plants with higher nutrient and lower PSM concentrations relative to other plants. Pygmy rabbits (Brachylagus idahoensis) are dietary specialists that feed on sagebrush (Artemisia spp.) and forage on specific plants more than others within a foraging patch. We predicted that the plants with evidence of heavy foraging (browsed plants) would be of higher dietary quality than plants that were not browsed (unbrowsed). We used model selection to determine which phytochemical variables best explained the difference between browsed and unbrowsed plants. Higher crude protein increased the odds that plants would be browsed by pygmy rabbits and the opposite was the case for certain PSMs. Additionally, because pygmy rabbits can occupy foraging patches (burrows) for consecutive years, their browsing may influence the nutritional and PSM constituents of plants at the burrows. In a post hoc analysis, we did not find a significant relationship between phytochemical concentrations, browse status and burrow occupancy length. We concluded that pygmy rabbits use nutritional and chemical cues while making foraging decisions. PMID:26366011

  5. Effect of soy protein on swine intestinal lipoproteins

    International Nuclear Information System (INIS)

    Ho, H.T.

    1987-01-01

    Hypocholesterolemic effect of soy protein appears to be the result of reduced cholesterol absorption and enhanced cholesterol excretion. The objective of this study is to delineate the underlying mechanism of soy protein effect on cholesterol absorption. At the end of a 5-week soy-protein or casein diet, swine were subjected to cannulation of mesenteric lymph duct under halothane anesthesia. A single dose of 250 μCi [ 14 C]-cholesterol and 10 mCi [ 3 H]-leucine was infused into the upper jejunum two hours after one-fifth of daily food was given. Then lymph was collected hourly for three hours and the lipoprotein fractions were separated by ultracentrifugation. SDS-PAGE (5%) was used to measure the concentrations of individual apoproteins by densitometric scanning. The three-hour lymphatic transport of cholesterol in casein-fed swine was significantly higher than in those fed soy protein. Triglyceride transports were similar in two groups. The [ 3 H]-leucine incorporation study revealed that transport of apo B-48 bore a significant positive relationship to transport of cholesterol in both chylomicron and VLDL fractions of mesenteric lymph. A greater apo B-48 secretion with higher specific activity was probably responsible for the greater transport of cholesterol in chylomicrons in casein-fed swine. On the other hand, the lesser cholesterol transport in chylomicrons in soy protein-fed swine was probably caused by lower apo B-48 secretion. Similarly, the transport of lymph VLDL cholesterol in swine fed casein or soy protein paralleled the amount of accompanying apo B-48. Dietary proteins probably influence the intestinal synthesis of apo B-48 which in turn affects cholesterol transport into the lymphatics

  6. Corrosion-product inventory: the Bruce-B secondary system

    International Nuclear Information System (INIS)

    Sawicki, J.A.; Price, J.; Brett, M.E.

    1995-01-01

    Corrosion inspection and corrosion-product characterization in water and steam systems are important for component and systems maintenance in nuclear power stations. Corrosion products are produced, released and redeposited at various sites in the secondary system. Depending on the alloys used in the condenser and feedwater heaters, particulate iron oxides and hydroxides can account for about 95-99% of the total corrosion-product transport. Where brass or cupro-nickel alloys are present, copper and zinc contribute significantly to the total transport and deposition. Particulates are transported by the feedwater to the steam generators, where they accumulate and can cause a variety of problems, such as loss of heat transfer capability through deposition on boiler tubes, blockage of flow through boiler-tube support plates and accelerated corrosion in crevices, either in deep sludge piles or at blocked tube supports. The influx of oxidized corrosion products may have a particularly adverse effect on the redox environment of steam generator tubing, thereby increasing the probability of localized corrosion and other degradation mechanisms. In this paper, there is a description of a survey of general corrosion deposits in Bruce-B, Units 5-8, which helps to identify the origin, evolution and inventory of corrosion products along the secondary system of Candu reactors

  7. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.

    Science.gov (United States)

    Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2006-11-01

    APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.

  8. Recent advances on uric acid transporters

    Science.gov (United States)

    Xu, Liuqing; Shi, Yingfeng; Zhuang, Shougang; Liu, Na

    2017-01-01

    Uric acid is the product of purine metabolism and its increased levels result in hyperuricemia. A number of epidemiological reports link hyperuricemia with multiple disorders, such as kidney diseases, cardiovascular diseases and diabetes. Recent studies also showed that expression and functional changes of urate transporters are associated with hyperuricemia. Uric acid transporters are divided into two categories: urate reabsorption transporters, including urate anion transporter 1 (URAT1), organic anion transporter 4 (OAT4) and glucose transporter 9 (GLUT9), and urate excretion transporetrs, including OAT1, OAT3, urate transporter (UAT), multidrug resistance protein 4 (MRP4/ABCC4), ABCG-2 and sodium-dependent phosphate transport protein. In the kidney, uric acid transporters decrease the reabsorption of urate and increase its secretion. These transporters’ dysfunction would lead to hyperuricemia. As the function of urate transporters is important to control the level of serum uric acid, studies on the functional role of uric acid transporter may provide a new strategy to treat hyperuricemia associated diseases, such as gout, chronic kidney disease, hyperlipidemia, hypertension, coronary heart disease, diabetes and other disorders. This review article summarizes the physiology of urate reabsorption and excretion transporters and highlights the recent advances on their roles in hyperuricemia and various diseases. PMID:29246027

  9. A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

    Science.gov (United States)

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

    2016-07-01

    TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. The Purine Bias of Coding Sequences is Determined by Physicochemical Constraints on Proteins.

    Science.gov (United States)

    Ponce de Leon, Miguel; de Miranda, Antonio Basilio; Alvarez-Valin, Fernando; Carels, Nicolas

    2014-01-01

    For this report, we analyzed protein secondary structures in relation to the statistics of three nucleotide codon positions. The purpose of this investigation was to find which properties of the ribosome, tRNA or protein level, could explain the purine bias (Rrr) as it is observed in coding DNA. We found that the Rrr pattern is the consequence of a regularity (the codon structure) resulting from physicochemical constraints on proteins and thermodynamic constraints on ribosomal machinery. The physicochemical constraints on proteins mainly come from the hydropathy and molecular weight (MW) of secondary structures as well as the energy cost of amino acid synthesis. These constraints appear through a network of statistical correlations, such as (i) the cost of amino acid synthesis, which is in favor of a higher level of guanine in the first codon position, (ii) the constructive contribution of hydropathy alternation in proteins, (iii) the spatial organization of secondary structure in proteins according to solvent accessibility, (iv) the spatial organization of secondary structure according to amino acid hydropathy, (v) the statistical correlation of MW with protein secondary structures and their overall hydropathy, (vi) the statistical correlation of thymine in the second codon position with hydropathy and the energy cost of amino acid synthesis, and (vii) the statistical correlation of adenine in the second codon position with amino acid complexity and the MW of secondary protein structures. Amino acid physicochemical properties and functional constraints on proteins constitute a code that is translated into a purine bias within the coding DNA via tRNAs. In that sense, the Rrr pattern within coding DNA is the effect of information transfer on nucleotide composition from protein to DNA by selection according to the codon positions. Thus, coding DNA structure and ribosomal machinery co-evolved to minimize the energy cost of protein coding given the functional

  11. ProteinShop: A tool for interactive protein manipulation and steering

    Energy Technology Data Exchange (ETDEWEB)

    Crivelli, Silvia; Kreylos, Oliver; Max, Nelson; Hamann, Bernd; Bethel, Wes

    2004-05-25

    We describe ProteinShop, a new visualization tool that streamlines and simplifies the process of determining optimal protein folds. ProteinShop may be used at different stages of a protein structure prediction process. First, it can create protein configurations containing secondary structures specified by the user. Second, it can interactively manipulate protein fragments to achieve desired folds by adjusting the dihedral angles of selected coil regions using an Inverse Kinematics method. Last, it serves as a visual framework to monitor and steer a protein structure prediction process that may be running on a remote machine. ProteinShop was used to create initial configurations for a protein structure prediction method developed by a team that competed in CASP5. ProteinShop's use accelerated the process of generating initial configurations, reducing the time required from days to hours. This paper describes the structure of ProteinShop and discusses its main features.

  12. Solute carrier transporters: Pharmacogenomics research ...

    African Journals Online (AJOL)

    Aghogho

    2010-12-27

    Dec 27, 2010 ... This paper reviews the solute carrier transporters and highlights the fact that there is much to be learnt from .... transporters, drug targets, effect or proteins and meta- ... basolateral or apical plasma membrane of polarized cells,.

  13. Regulators of Slc4 bicarbonate transporter activity

    Directory of Open Access Journals (Sweden)

    Ian M. Thornell

    2015-06-01

    Full Text Available The Slc4 family of transporters is comprised of anion exchangers (AE1-4, Na-coupled bicarbonate transporters (NCBTs including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2, electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2, and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE, as well as a borate transporter (BTR1. These transporters regulate intracellular pH (pHi and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO3– either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO3– transporter contributes to a cell’s ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s (e.g., Na+ or Cl–. In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both classical and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family.

  14. The rise and fall of novel renal magnesium transporters.

    Science.gov (United States)

    Schäffers, Olivier J M; Hoenderop, Joost G J; Bindels, René J M; de Baaij, Jeroen H F

    2018-06-01

    Body Mg 2+ balance is finely regulated in the distal convoluted tubule (DCT), where a tight interplay among transcellular reabsorption, mitochondrial exchange, and basolateral extrusion takes place. In the last decades, several research groups have aimed to identify the molecular players in these processes. A multitude of proteins have been proposed to function as Mg 2+ transporter in eukaryotes based on phylogenetic analysis, differential gene expression, and overexpression studies. However, functional evidence for many of these proteins is lacking. The aim of this review is, therefore, to critically reconsider all putative Mg 2+ transporters and put their presumed function in context of the renal handling of Mg 2+ . Sufficient experimental evidence exists to acknowledge transient receptor potential melastatin (TRPM) 6 and TRPM7, solute carrier family 41 (SLC41) A1 and SLC41A3, and mitochondrial RNA splicing 2 (MRS2) as Mg 2+ transporters. TRPM6/7 facilitate Mg 2+ influx, SLC41A1 mediates Mg 2+ extrusion, and MRS2 and SLC41A3 are implicated in mitochondrial Mg 2+ homeostasis. These proteins are highly expressed in the DCT. The function of cyclin M (CNNM) proteins is still under debate. For the other proposed Mg 2+ transporters including Mg 2+ transporter subtype 1 (MagT1), nonimprinted in Prader-Willi/Angelman syndrome (NIPA), membrane Mg 2+ transport (MMgT), Huntingtin-interacting protein 14 (HIP14), and ATP13A4, functional evidence is limited, or functions alternative to Mg 2+ transport have been suggested. Additional characterization of their Mg 2+ transport proficiency should be provided before further claims about their role as Mg 2+ transporter can be made.

  15. Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters

    International Nuclear Information System (INIS)

    Straka, Elisabeth; Ellinger, Isabella; Balthasar, Christina; Scheinast, Matthias; Schatz, Jasmin; Szattler, Tamara; Bleichert, Sonja; Saleh, Leila; Knöfler, Martin; Zeisler, Harald; Hengstschläger, Markus; Rosner, Margit; Salzer, Hans; Gundacker, Claudia

    2016-01-01

    Highlights: • It is known that MeHg is able to pass the placenta and to affect fetal brain development. • Uptake and efflux transporters were examined in human primary trophoblast cells and BeWo cells. • Involvement in mercury transfer was assessed by measurement of cellular mercury content upon siRNA mediated gene knockdown. • Localization of transporters was determined by immunofluorescence microscopy. • LAT1 and rBAT at the apical membrane of the syncytiotrophoblast (STB) are involved in MeHg uptake. • MRP1 located at basal membrane of STB mediates mercury efflux. - Abstract: Background: The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. Objectives: Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. Methods: To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by Cold vapor-atomic fluorescence spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. Results: We found the amino acid transporter subunits L-type amino acid transporter (LAT)1 and rBAT (related to b 0,+ type amino acid transporter) as well as the efflux transporter multidrug resistance associated protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-test P < 0.05). Conclusion: The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is

  16. Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes

    International Nuclear Information System (INIS)

    Rivera, Alicia; De Franceschi, Lucia; Peters, Luanne L.; Gascard, Philippe; Mohandas, Narla; Brugnara, Carlo

    2006-01-01

    Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1-/-; Shi TS et al., J. Clin. Invest. 103:331,1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1-/- mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1-/- erythrocytes was markedly activated by exposure to hypertonic conditions (18.2+- 3.2 in 4.1 -/- vs.9.8 +- 1.3 mmol/1013 cell x h in control mice), with an abnormal dependence on osmolarity, (K0.5=417 +- 42 in 4.1 -/- vs. 460 +- 35 mOsmin control mice) suggestive of an up-regulated functional state. While the affinity for internal protons was not altered (K0.5= 489.7 +- 0.7 vs.537.0 +- 0.56 nM in control mice), the Vmax of the H-induced Na/H exchange activity was markedly elevated in 4.1-/- erythrocytes Vmax 91.47 Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1-/-; Shi TSet al., J. Clin. Invest. 103:331,1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1-/- mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1-/- erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 +- 3.2 in 4.1 -/- vs. 9.8 +- 1.3mmol/1013 cell x h in

  17. A nu-space for image correlation spectroscopy: characterization and application to measure protein transport in live cells

    Science.gov (United States)

    Potvin-Trottier, Laurent; Chen, Lingfeng; Horwitz, Alan Rick; Wiseman, Paul W.

    2013-08-01

    We introduce a new generalized theoretical framework for image correlation spectroscopy (ICS). Using this framework, we extend the ICS method in time-frequency (ν, nu) space to map molecular flow of fluorescently tagged proteins in individual living cells. Even in the presence of a dominant immobile population of fluorescent molecules, nu-space ICS (nICS) provides an unbiased velocity measurement, as well as the diffusion coefficient of the flow, without requiring filtering. We also develop and characterize a tunable frequency-filter for spatio-temporal ICS (STICS) that allows quantification of the density, the diffusion coefficient and the velocity of biased diffusion. We show that the techniques are accurate over a wide range of parameter space in computer simulation. We then characterize the retrograde flow of adhesion proteins (α6- and αLβ2-GFP integrins and mCherry-paxillin) in CHO.B2 cells plated on laminin and intercellular adhesion molecule 1 (ICAM-1) ligands respectively. STICS with a tunable frequency filter, in conjunction with nICS, measures two new transport parameters, the density and transport bias coefficient (a measure of the diffusive character of a flow/biased diffusion), showing that molecular flow in this cell system has a significant diffusive component. Our results suggest that the integrin-ligand interaction, along with the internal myosin-motor generated force, varies for different integrin-ligand pairs, consistent with previous results.

  18. Legionella pneumophila secretes a mitochondrial carrier protein during infection.

    Directory of Open Access Journals (Sweden)

    Pavel Dolezal

    2012-01-01

    Full Text Available The Mitochondrial Carrier Family (MCF is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP, encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

  19. Secondary-electron cascade in attosecond photoelectron spectroscopy from metals

    DEFF Research Database (Denmark)

    Baggesen, Jan Conrad; Madsen, Lars Bojer

    2009-01-01

    an analytical model based on an approximate solution to Boltzmann's transport equation to account for the amount and energy distribution of these secondary electrons. Our theory is in good agreement with the electron spectrum found in a recent attosecond streaking experiment. To suppress the background and gain......Attosecond spectroscopy is currently restricted to photon energies around 100 eV. We show that under these conditions, electron-electron scatterings, as the photoelectrons leave the metal, give rise to a tail of secondary electrons with lower energies and hence a significant background. We develop...

  20. Unraveling the meaning of chemical shifts in protein NMR.

    Science.gov (United States)

    Berjanskii, Mark V; Wishart, David S

    2017-11-01

    Chemical shifts are among the most informative parameters in protein NMR. They provide wealth of information about protein secondary and tertiary structure, protein flexibility, and protein-ligand binding. In this report, we review the progress in interpreting and utilizing protein chemical shifts that has occurred over the past 25years, with a particular focus on the large body of work arising from our group and other Canadian NMR laboratories. More specifically, this review focuses on describing, assessing, and providing some historical context for various chemical shift-based methods to: (1) determine protein secondary and super-secondary structure; (2) derive protein torsion angles; (3) assess protein flexibility; (4) predict residue accessible surface area; (5) refine 3D protein structures; (6) determine 3D protein structures and (7) characterize intrinsically disordered proteins. This review also briefly covers some of the methods that we previously developed to predict chemical shifts from 3D protein structures and/or protein sequence data. It is hoped that this review will help to increase awareness of the considerable utility of NMR chemical shifts in structural biology and facilitate more widespread adoption of chemical-shift based methods by the NMR spectroscopists, structural biologists, protein biophysicists, and biochemists worldwide. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

    Science.gov (United States)

    Chambouvet, Aurélie; Milner, David S.; Attah, Victoria; Terrado, Ramón; Lovejoy, Connie; Moreau, Hervé; Derelle, Évelyne; Richards, Thomas A.

    2017-01-01

    Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer. PMID:28827361

  2. A comparative method for finding and folding RNA secondary structures within protein-coding regions

    DEFF Research Database (Denmark)

    Pedersen, Jakob Skou; Meyer, Irmtraud Margret; Forsberg, Roald

    2004-01-01

    that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known...... secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD....

  3. Identification and characterization of a novel Cut family cDNA that encodes human copper transporter protein CutC

    International Nuclear Information System (INIS)

    Li Jixi; Ji Chaoneng; Chen Jinzhong; Yang Zhenxing; Wang Yijing; Fei, Xiangwei; Zheng Mei; Gu Xing; Wen Ge; Xie Yi; Mao Yumin

    2005-01-01

    Copper is an essential heavy metal trace element that plays important roles in cell physiology. The Cut family was associated with the copper homeostasis and involved in several important metabolisms, such as uptake, storage, delivery, and efflux of copper. In this study, a novel Cut family cDNA was isolated from the human fetal brain library, which encodes a 273 amino acid protein with a molecular mass of about 29.3 kDa and a calculated pI of 8.17. It was named hCutC (human copper transporter protein CutC). The ORF of hCutC gene was cloned into pQE30 vector and expressed in Escherichia coli M15. The secreted hCutC protein was purified to a homogenicity of 95% by using the Ni-NTA affinity chromatography. RT-PCR analysis showed that the hCutC gene expressed extensively in human tissues. Subcellular location analysis of hCutC-EGFP fusion protein revealed that hCutC was distributed to cytoplasm of COS-7 cells, and both cytoplasm and nucleus of AD293 cells. The results suggest that hCutC may be one shuttle protein and play important roles in intracellular copper trafficking

  4. Variability of Atmospheric Radon-222 and Secondary Aerosol Components in Accordance with Air Mass Transport Pathways at Jeju Island, Korea, during 2011-2014

    International Nuclear Information System (INIS)

    Bu, Jun-Oh; Song, Jung-Min; Kim, Won-Hyung; Kang, Chang-Hee; Chambers, Scott D.; Williams, Alastair G.; Lee, Chulkyu

    2016-01-01

    Real-time monitoring of hourly atmospheric Radon-222 concentration and three daily monitoring of the secondary aerosol components of PM_1_0 were performed throughout 2011-2014 at Gosan station, Jeju Island, in order to characterize their background levels and temporal variation. The annual mean radon and PM_1_0 mass concentrations were 2326 ± 1198 mBq/m"3 and 37.1 ± 19.5 μg/m"3, respectively. Based on cluster analyses of air mass back trajectories, the frequencies of air masses originating from continental China, the Korean Peninsula, and North Pacific Ocean routes were 53, 28, and 19%, respectively. When the air masses were transported to Jeju Island from continental China, the concentrations of radon and secondary aerosol components (nss-SO_4"2"-, NO_3"-, NH_4"+) were relatively high: 2577 mBq/m"3 and 14.4 μg/m"3, respectively. In cases when the air masses have moved from the Korean Peninsula, the corresponding concentrations were 2247 mBq/m"3 and 11.4 μg/m"3, respectively. On the other hand, when the air masses came from the North Pacific Ocean, their radon and secondary aerosol concentrations decreased much further, 1372 mBq/m"3 and 10.5 μg/m"3, respectively. Consequently, the variability of atmospheric radon concentrations at Gosan station might be characterized by synoptic changes in air mass fetch as well as diurnal changes in atmospheric mixing depth.

  5. Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life

    DEFF Research Database (Denmark)

    Bøttger, Pernille; Pedersen, Lene

    2011-01-01

    -keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian Pi...... PiT2 histidine, H502, and the human PiT1 glutamate, E70, - both conserved in eukaryotic PiT family members - are critical for Pi transport function. Noticeably, human PiT2 H502 is located in the C-terminal PiT family signature sequence, and human PiT1 E70 is located in ProDom domains characteristic....... Conclusions The results suggest that the overall structure of the Pi-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein...

  6. Potassium-transporting proteins in skeletal muscle: cellular location and fiber-type differences

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten

    2010-01-01

    Potassium (K+) displacement in skeletal muscle may be an important factor in the development of muscle fatigue during intense exercise. It has been shown in vitro that an increase in the extracellular K+ concentration ([K+]e) to values higher than approx. 10 mm significantly reduce force developm......Potassium (K+) displacement in skeletal muscle may be an important factor in the development of muscle fatigue during intense exercise. It has been shown in vitro that an increase in the extracellular K+ concentration ([K+]e) to values higher than approx. 10 mm significantly reduce force......, but is suggested primarily to participate in K+ release to the interstitium. Because there is restricted diffusion of K+ to the interstitium, K+ released to the T-tubules during AP propagation will be removed primarily by reuptake mediated by transport proteins located in the T-tubule membrane. The most important...

  7. Role of transporters in placental transfer of drugs

    International Nuclear Information System (INIS)

    Ganapathy, Vadivel; Prasad, Puttur D.

    2005-01-01

    Human placenta functions as an important transport organ that mediates the exchange of nutrients and metabolites between maternal and fetal circulations. This function is made possible because of the expression of a multitude of transport proteins in the placental syncytiotrophoblast with differential localization in the maternal-facing brush border membrane versus the fetal-facing basal membrane. Even though the physiological role of most of these transport proteins is to handle nutrients, many of them interact with xenobiotics and pharmacological agents. These transport proteins therefore play a critical role in the disposition of drugs across the maternal-fetal interface, with some transporters facilitating the entry of drugs from maternal circulation into fetal circulation whereas others preventing such entry by actively eliminating drugs from the placenta back into maternal circulation. The net result as to whether the placenta enhances the exposure of the developing fetus to drugs and xenobiotics or functions as a barrier to protect the fetus from such agents depends on the types of transporters expressed in the brush border membrane and basal membrane of the syncytiotrophoblast and on the functional mode of these transporters (influx versus efflux)

  8. The contribution of transport to air quality. TERM 2012: transport indicators tracking progress towards environmental targets in Europe

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez Vicente, A.; Pastorello, C.; Foltescu, V.L. [and others

    2012-11-15

    TERM 2012 (Transport and Environment Reporting Mechanism) presents the most relevant and up to date information on the main issues regarding transport and environment in Europe, particularly in areas with specific policy targets such as greenhouse gas emissions and energy consumption, transport demand levels, noise and other issues. It also offers an overview of the transport sector's impact on air pollutant emissions and air quality. It discusses the contributions made by all modes of transport to direct air pollutant emissions and also to 'secondary' air pollutants formed in the atmosphere. Alongside the recently published Air quality in Europe - 2012 report, TERM 2012 aims to inform the European Commission's review of the Thematic Strategy on Air Pollution. (Author)

  9. A Glimpse of Membrane Transport through Structures-Advances in the Structural Biology of the GLUT Glucose Transporters.

    Science.gov (United States)

    Yan, Nieng

    2017-08-18

    The cellular uptake of glucose is an essential physiological process, and movement of glucose across biological membranes requires specialized transporters. The major facilitator superfamily glucose transporters GLUTs, encoded by the SLC2A genes, have been a paradigm for functional, mechanistic, and structural understanding of solute transport in the past century. This review starts with a glimpse into the structural biology of membrane proteins and particularly membrane transport proteins, enumerating the landmark structures in the past 25years. The recent breakthrough in the structural elucidation of GLUTs is then elaborated following a brief overview of the research history of these archetypal transporters, their functional specificity, and physiological and pathophysiological significances. Structures of GLUT1, GLUT3, and GLUT5 in distinct transport and/or ligand-binding states reveal detailed mechanisms of the alternating access transport cycle and substrate recognition, and thus illuminate a path by which structure-based drug design may be applied to help discover novel therapeutics against several debilitating human diseases associated with GLUT malfunction and/or misregulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

    Science.gov (United States)

    Moya, K L; Confaloni, A M; Allinquant, B

    1994-11-01

    We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

  11. Divide and conquer: processive transport enables multidrug transporters to tackle challenging drugs

    Directory of Open Access Journals (Sweden)

    Nir Fluman

    2014-09-01

    Full Text Available Multidrug transporters are membrane proteins that catalyze efflux of antibiotics and other toxic compounds from cells, thereby conferring drug resistance on various organisms. Unlike most solute transporters that transport a single type of compound or similar analogues, multidrug transporters are extremely promiscuous. They transport a broad spectrum of dissimilar drugs and represent a serious obstacle to antimicrobial or anticancer chemotherapy. Many challenging aspects of multidrug transporters, which are unique, have been studied in detail, including their ability to interact with chemically unrelated drugs, and how they utilize energy to drive efflux of compounds that are not only structurally but electrically different. A new and surprising dimension of the promiscuous nature of multidrug transporters has been described recently: they can move long molecules through the membrane in a processive manner.

  12. Temporal resolution of misfolded prion protein transport, accumulation, glial activation, and neuronal death in the retinas of mice inoculated with scrapie

    Science.gov (United States)

    Currently, there is a lack of pathologic landmarks to describe the progression of prion disease in vivo. The goal of this work was to determine the temporal relationship between the transport of misfolded prion protein from the brain to the retina, the accumulation of PrPSc in the retina, the respon...

  13. Mutational Analysis on Membrane Associated Transporter Protein (MATP) and Their Structural Consequences in Oculocutaeous Albinism Type 4 (OCA4)-A Molecular Dynamics Approach.

    Science.gov (United States)

    Kamaraj, Balu; Purohit, Rituraj

    2016-11-01

    Oculocutaneous albinism type IV (OCA4) is an autosomal recessive inherited disorder which is characterized by reduced biosynthesis of melanin pigmentation in skin, hair, and eyes and caused by the genetic mutations in the membrane-associated transporter protein (MATP) encoded by SLC45A2 gene. The MATP protein consists of 530 amino acids which contains 12 putative transmembrane domains and plays an important role in pigmentation and probably functions as a membrane transporter in melanosomes. We scrutinized the most OCA4 disease-associated mutation and their structural consequences on SLC45A2 gene. To understand the atomic arrangement in 3D space, the native and mutant structures were modeled. Further the structural behavior of native and mutant MATP protein was investigated by molecular dynamics simulation (MDS) approach in explicit lipid and water background. We found Y317C as the most deleterious and disease-associated SNP on SLC45A2 gene. In MDS, mutations in MATP protein showed loss of stability and became more flexible, which alter its structural conformation and function. This phenomenon has indicated a significant role in inducing OCA4. Our study explored the understanding of molecular mechanism of MATP protein upon mutation at atomic level and further helps in the field of pharmacogenomics to develop a personalized medicine for OCA4 disorder. J. Cell. Biochem. 117: 2608-2619, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Hyoun-Sub, E-mail: hyounlim@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Nam, Jiryun, E-mail: jilyoon@naver.com [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Seo, Eun-Young, E-mail: sey22@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Nam, Moon, E-mail: moonlit51@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Vaira, Anna Maria, E-mail: a.vaira@ivv.cnr.it [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States); Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino 10135 (Italy); Bae, Hanhong, E-mail: hanhongbae@ynu.ac.kr [School of Biotechnology, Yeungnam University, Geongsan 712-749 (Korea, Republic of); Jang, Chan-Yong, E-mail: sunbispirit@gmail.com [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Cheol Ho, E-mail: chlee1219@hanmail.net [Department of Chemical and Biological Engineering, Seokyoung University, Seoul 136-704 (Korea, Republic of); Kim, Hong Gi, E-mail: hgkim@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Roh, Mark, E-mail: marksroh@gmail.com [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States); Laboratory of Floriculture and Plant Physiology, School of Bio-Resource Science, Dankook University, Cheonan, Chungnam 330-714 (Korea, Republic of); Hammond, John, E-mail: john.hammond@ars.usda.gov [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States)

    2014-03-15

    Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP{sub SP}) with that from AltMV-Po (CP{sub Po}) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP{sub Po} [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP{sub SP} but not CP{sub Po} interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP{sub SP} than CP{sub Po} in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. - Highlights: • Alternanthera mosaic virus CP is an elicitor of systemic necrosis in N. benthamiana. • Virus-induced systemic necrosis is enhanced at 15 °C compared to 25 °C. • Induction of systemic necrosis is dependent on as few as two CP amino acid residues. • These residues are at subunit interfaces within the same turn of the virion helix. • Inducer/non-inducer CPs interact differentially with a boron transporter protein.

  15. Assessment of secondary sources of Persistent Organic Pollutants in the Arctic

    Science.gov (United States)

    Pisso, Ignacio; Eckhardt, Sabine; Breivik, Knut

    2014-05-01

    Persistent organic pollutants (POPs) including highly toxic pesticides and other chemicals accumulate in living tissues and magnify in food chains. POPs are subject to long-range transport and hence represent a serious public health issue even in regions where their production is regulated. Rational control strategies require an understanding of the overall relationship between environmental emissions of contaminants and environmental / human exposure. In this study, we assess the relationships between environmental emissions and potential human exposure of organic contaminants with emphasis on long-range atmospheric transport. We investigate whether atmospheric levels of POPs measured at Zeppelin observatory in Svalbard since the early '90s are controlled by primary or secondary emissions. We present statistical indications that the measurements are affected by secondary ocean emissions and discuss the applicability of different inverse modeling approaches.

  16. Regulation of ER-Golgi Transport Dynamics by GTPases in Budding Yeast

    Directory of Open Access Journals (Sweden)

    Yasuyuki Suda

    2018-01-01

    Full Text Available A large number of proteins are synthesized de novo in the endoplasmic reticulum (ER. They are transported through the Golgi apparatus and then delivered to their proper destinations. The ER and the Golgi play a central role in protein processing and sorting and show dynamic features in their forms. Ras super family small GTPases mediate the protein transport through and between these organelles. The ER-localized GTPase, Sar1, facilitates the formation of COPII transport carriers at the ER exit sites (ERES on the ER for the transport of cargo proteins from the ER to the Golgi. The Golgi-localized GTPase, Arf1, controls intra-Golgi, and Golgi-to-ER transport of cargo proteins by the formation of COPI carriers. Rab GTPases localized at the Golgi, which are responsible for fusion of membranes, are thought to establish the identities of compartments. Recent evidence suggests that these small GTPases regulate not only discrete sites for generation/fusion of transport carriers, but also membrane dynamics of the organelles where they locate to ensure the integrity of transport. Here we summarize the current understandings about the membrane traffic between these organelles and highlight the cutting-edge advances from super-resolution live imaging of budding yeast, Saccharomyces cerevisiae.

  17. Nestin upregulation characterizes vascular remodeling secondary to hypertension in the rat.

    Science.gov (United States)

    Tardif, Kim; Hertig, Vanessa; Duquette, Natacha; Villeneuve, Louis; El-Hamamsy, Ismail; Tanguay, Jean-François; Calderone, Angelino

    2015-05-15

    Proliferation and hypertrophy of vascular smooth muscle cells represent hallmark features of vessel remodeling secondary to hypertension. The intermediate filament protein nestin was recently identified in vascular smooth muscle cells and in other cell types directly participated in proliferation. The present study tested the hypothesis that vessel remodeling secondary to hypertension was characterized by nestin upregulation in vascular smooth muscle cells. Two weeks after suprarenal abdominal aorta constriction of adult male Sprague-Dawley rats, elevated mean arterial pressure increased the media area and thickness of the carotid artery and aorta and concomitantly upregulated nestin protein levels. In the normal adult rat carotid artery, nestin immunoreactivity was observed in a subpopulation of vascular smooth muscle cells, and the density significantly increased following suprarenal abdominal aorta constriction. Filamentous nestin was detected in cultured rat carotid artery- and aorta-derived vascular smooth muscle cells and an analogous paradigm observed in human aorta-derived vascular smooth muscle cells. ANG II and EGF treatment of vascular smooth muscle cells stimulated DNA and protein synthesis and increased nestin protein levels. Lentiviral short-hairpin RNA-mediated nestin depletion of carotid artery-derived vascular smooth muscle cells inhibited peptide growth factor-stimulated DNA synthesis, whereas protein synthesis remained intact. These data have demonstrated that vessel remodeling secondary to hypertension was characterized in part by nestin upregulation in vascular smooth muscle cells. The selective role of nestin in peptide growth factor-stimulated DNA synthesis has revealed that the proliferative and hypertrophic responses of vascular smooth muscle cells were mediated by divergent signaling events. Copyright © 2015 the American Physiological Society.

  18. Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation

    Directory of Open Access Journals (Sweden)

    Kouki eYoshida

    2013-10-01

    Full Text Available Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs can regulate secondary wall formation in rice (Oryza sativa and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S has very low transcriptional activation ability, but the longer protein (OsSWN2L and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

  19. Monte Carlo simulation of positron induced secondary electrons in thin carbon foils

    International Nuclear Information System (INIS)

    Cai, L H; Yang, B; Ling, C C; Beling, C D; Fung, S

    2011-01-01

    Emission of secondary electrons induced by the passage of low energy positrons through thin carbon foils was studied by the Monte Carlo method. The positron and electron elastic cross sections were calculated by partial wave analysis. The inelastic positron-valence-electron was described by the energy loss function obtained from dielectric theory. The positron-core-electron interaction was modelled by the Gryzinski's excitation function. Positron transport inside the carbon foil was simulated in detail. Secondary electrons created by positrons and high energy secondary electrons through inelastic interactions were tracked through the foil. The positron transmission coefficient and secondary electron yielded in forward and backward geometry are calculated and dependences on positron energy and carbon foil thickness are discussed.

  20. Molecular mechanisms of water transport in the eye

    DEFF Research Database (Denmark)

    Hamann, Steffen

    2002-01-01

    The four major sites for ocular water transport, the corneal epithelium and endothelium, the ciliary epithelium, and the retinal pigment epithelium, are reviewed. The cornea has an inherent tendency to swell, which is counteracted by its two surface cell layers, the corneal epithelium...... and endothelium. The bilayered ciliary epithelium secretes the aqueous humor into the posterior chamber, and the retinal pigment epithelium transports water from the retinal to the choroidal site. For each epithelium, ion transport mechanisms are associated with fluid transport, but the exact molecular coupling...... sites between ion and water transport remain undefined. In the retinal pigment epithelium, a H+-lactate cotransporter transports water. This protein could be the site of coupling between salt and water in this epithelium. The distribution of aquaporins does not suggest a role for these proteins...