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Sample records for high-affinity sucrose transporter

  1. A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

    PubMed Central

    Goos, Sarah; Kämper, Jörg; Sauer, Norbert

    2010-01-01

    Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host. PMID:20161717

  2. High-affinity ammonium transporters and nitrogen sensing in mycorrhizas.

    PubMed

    Javelle, Arnaud; André, Bruno; Marini, Anne Marie; Chalot, Michel

    2003-02-01

    Most terrestrial plants live in mutualistic symbiosis with root-infecting mycorrhizal fungi. This association requires a molecular dialogue between the two partners. However, the nature of the chemical signals that induce hyphal differentiation are not well characterized and the mechanisms for signal reception are still unknown. In addition to its role in ammonium scavenging, the Mep2 protein from Saccharomyces cerevisiae has been proposed to act as an ammonium sensor that is essential for pseudohyphal differentiation in response to ammonium limitation. We propose that the high-affinity ammonium transporters from mycorrhizal fungi act in a similar manner to sense the environment and induce, via as-yet-unidentified signal transduction cascades, the switch in the mode of fungal growth observed during the formation of mycorrhiza.

  3. Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2004-10-01

    High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots. Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively. Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+.

  4. Expression Cloning of the High Affinity Choline Transporter

    DTIC Science & Technology

    1993-05-05

    clones. It encodes a GABA transporter that we found to be localized to the glial cells of the purely cholinergic electromotor nucleus of Torpedo. In a...expression cloning approach employing frog oocytes and mRNA from Torpedo C.B. Gundersen electromotor nucleus to isolate a choline transporter cDNA...The rationale for this is that the electromotor neurons should harbor one of the highest abundances of choline transporter mRNA in the animal kingdom

  5. Mathematical modeling of the low and high affinity arabinose transport systems in Escherichia coli.

    PubMed

    Yildirim, Necmettin

    2012-04-01

    A mathematical model was developed for the low and high affinity arabinose transport systems in E. coli. The model is a system of three ordinary differential equations and takes the dynamics of mRNAs for the araE and araFGH proteins and the internal arabinose into account. Special attention was paid to estimate the model parameters from the literature. Our analysis and simulations suggest that the high affinity transport system helps the low affinity transport system to respond to high concentration of extracellular arabinose faster, whereas the high affinity transport system responds to a small amount of extracellular arabinose. Steady state analysis of the model also predicts that there is a regime for the extracellular concentration of arabinose where the arabinose system can show bistable behavior.

  6. Functional transformations of bile acid transporters induced by high-affinity macromolecules

    PubMed Central

    Al-Hilal, Taslim A.; Chung, Seung Woo; Alam, Farzana; Park, Jooho; Lee, Kyung Eun; Jeon, Hyesung; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, In-San; Kim, Sang Yoon; Byun, Youngro

    2014-01-01

    Apical sodium-dependent bile acid transporters (ASBT) are the intestinal transporters that form intermediate complexes with substrates and its conformational change drives the movement of substrates across the cell membrane. However, membrane-based intestinal transporters are confined to the transport of only small molecular substrates. Here, we propose a new strategy that uses high-affinity binding macromolecular substrates to functionally transform the membrane transporters so that they behave like receptors, ultimately allowing the apical-basal transport of bound macromolecules. Bile acid based macromolecular substrates were synthesized and allowed to interact with ASBT. ASBT/macromolecular substrate complexes were rapidly internalized in vesicles, localized in early endosomes, dissociated and escaped the vesicular transport while binding of cytoplasmic ileal bile acid binding proteins cause exocytosis of macromolecules and prevented entry into lysosomes. This newly found transformation process of ASBT suggests a new transport mechanism that could aid in further utilization of ASBT to mediate oral macromolecular drug delivery. PMID:24566561

  7. Pharmacological characterization of a high-affinity p-tyramine transporter in rat brain synaptosomes

    PubMed Central

    Berry, Mark D.; Hart, Shannon; Pryor, Anthony R.; Hunter, Samantha; Gardiner, Danielle

    2016-01-01

    p-Tyramine is an archetypal member of the endogenous family of monoamines known as trace amines, and is one of the endogenous agonists for trace amine-associated receptor (TAAR)1. While much work has focused on the function of TAAR1, very little is known about the regulation of the endogenous agonists. We have previously reported that p-tyramine readily crosses lipid bilayers and that its release from synaptosomes is non-exocytotic. Such release, however, showed characteristics of modification by one or more transporters. Here we provide the first characterization of such a transporter. Using frontal cortical and striatal synaptosomes we show that p-tyramine passage across synaptosome membranes is not modified by selective inhibition of either the dopamine, noradrenaline or 5-HT transporters. In contrast, inhibition of uptake-2 transporters significantly slowed p-tyramine re-uptake. Using inhibitors of varying selectivity, we identify Organic Cation Transporter 2 (OCT2; SLC22A2) as mediating high affinity uptake of p-tyramine at physiologically relevant concentrations. Further, we confirm the presence of OCT2 protein in synaptosomes. These results provide the first identification of a high affinity neuronal transporter for p-tyramine, and also confirm the recently described localization of OCT2 in pre-synaptic terminals. PMID:27901065

  8. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  9. High-affinity L-arabinose transport operon. Gene product expression and mRNAs.

    PubMed

    Horazdovsky, B F; Hogg, R W

    1987-09-05

    Various portions of the "high-affinity" L-arabinose transport operon were cloned into the plasmid expression vector pKK223-3 and the operon-encoded protein products were identified. The results indicate that three proteins are encoded by this operon. The first is a 33,000 Mr protein that is the product of the promoter-proximal L-arabinose binding protein coding sequence, araF. A 52,000 Mr protein is encoded by sequence 3' to araF and has been assigned to the araG locus. The sequence 3' to araG encodes a 31,000 Mr protein that has been assigned to the araH locus. Both the araG and araH gene products are localized in the membrane fraction of the cell, implying a role in the membrane-associated complex of the high-affinity L-arabinose transport system. Nuclease S1 protection studies indicate that two operon message populations are present in the cell, a full-length operon transcript and a seven- to tenfold more abundant binding protein-specific message. The relative abundance of these two message populations correlates with the differential expression of the binding protein and the membrane-associated proteins of the transport system.

  10. Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter

    PubMed Central

    Choudhary, Parul; Armstrong, Emma J.; Jorgensen, Csilla C.; Piotrowski, Mary; Barthmes, Maria; Torella, Rubben; Johnston, Sarah E.; Maruyama, Yuya; Janiszewski, John S.; Storer, R. Ian; Skerratt, Sarah E.; Benn, Caroline L.

    2017-01-01

    Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry. PMID:28289374

  11. Mechanism of high affinity inhibition of the human urate transporter URAT1

    PubMed Central

    Tan, Philip K.; Ostertag, Traci M.; Miner, Jeffrey N.

    2016-01-01

    Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions. PMID:27713539

  12. Functional Analysis of Arabidopsis Sucrose Transporters

    SciTech Connect

    John M. Ward

    2009-03-31

    Sucrose is the main photosynthetic product that is transported in the vasculature of plants. The long-distance transport of carbohydrates is required to support the growth and development of net-importing (sink) tissues such as fruit, seeds and roots. This project is focused on understanding the transport mechanism sucrose transporters (SUTs). These are proton-coupled sucrose uptake transporters (membrane proteins) that are required for transport of sucrose in the vasculature and uptake into sink tissues. The accomplishments of this project included: 1) the first analysis of substrate specificity for any SUT. This was accomplished using electrophysiology to analyze AtSUC2, a sucrose transporter from companion cells in Arabidopsis. 2) the first analysis of the transport activity for a monocot SUT. The transport kinetics and substrate specificity of HvSUT1 from barley were studied. 3) the first analysis of a sucrose transporter from sugarcane. and 4) the first analysis of transport activity of a sugar alcohol transporter homolog from plants, AtPLT5. During this period four primary research papers, funded directly by the project, were published in refereed journals. The characterization of several sucrose transporters was essential for the current effort in the analysis of structure/function for this gene family. In particular, the demonstration of strong differences in substrate specificity between type I and II SUTs was important to identify targets for site-directed mutagenesis.

  13. Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

    PubMed Central

    Cássio, F; Leáo, C

    1991-01-01

    Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1664712

  14. Substrate-induced internalization of the high-affinity choline transporter.

    PubMed

    Okuda, Takashi; Konishi, Asami; Misawa, Hidemi; Haga, Tatsuya

    2011-10-19

    Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

  15. Sucrose transport into stalk tissue of sugarcane

    SciTech Connect

    Thom, M.; Maretzki, A. )

    1990-05-01

    The productivity of higher plants is, in part, dependent on transport of photosynthate from source to sink (in sugarcane, stalk) and upon its assimilation in cells of the sink tissue. In sugarcane, sucrose has been reported to undergo hydrolysis in the apoplast before uptake into the storage parenchyma, whereas recently, sucrose was reported to be taken up intact. This work was based on lack of randomization of ({sup 14}C)fructosyl sucrose accumulated after feeding tissue slices with this sugar. In this report, we present evidence from slices of stalk tissue that sucrose is taken up intact via a carrier-mediated, energy-dependent process. The evidence includes: (1) uptake of fluorosucrose, an analog of sucrose not subject to hydrolysis by invertase; (2) little or no randomization of ({sup 14}C) fructosyl sucrose taken up; (3) the presence of a saturable as well as a linear component of sucrose uptake; and (4) inhibition of both the saturable and linear components of sucrose uptake by protonophore and sulhydryl agents. Hexoses can also be taken up, and at a greater efficiency than sucrose. It is probable that both hexose and sucrose can be transported across the plasma membrane, depending on the physiological status of the plant.

  16. The High-Affinity E. Coli Methionine ABC Transporter: Structure And Allosteric Regulation

    SciTech Connect

    Kadaba, N.S.; Kaiser, J.T.; Johnson, E.; Lee, A.; Rees, D.C.

    2009-05-18

    The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

  17. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-07-15

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins.

  18. Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.

    PubMed Central

    Cánovas, D; Vargas, C; Csonka, L N; Ventosa, A; Nieto, J J

    1996-01-01

    The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium. PMID:8955405

  19. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT

    PubMed Central

    Fishwick, Katherine J.; Rylett, R. Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer’s Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  20. High-affinity L-arabinose transport operon. Nucleotide sequence and analysis of gene products.

    PubMed

    Scripture, J B; Voelker, C; Miller, S; O'Donnell, R T; Polgar, L; Rade, J; Horazdovsky, B F; Hogg, R W

    1987-09-05

    The nucleotide sequence of the "high-affinity" L-arabinose transport operon has been determined 3' from the regulatory region and found to contain three open reading frames designated araF, araG and araH. The first gene 3' to the regulatory region, araF, encodes the 23-residue signal peptide and the 306-residue mature form of the L-arabinose binding protein (33,200 Mr). The binding protein, which has been described elsewhere, is hydrophilic, soluble and found in the periplasm of Escherichia coli. This gene is followed by an intragenic space of 72 nucleotides, which contains a region of dyad symmetry 23 nucleotides long capable of forming an 11-member stem-loop. The second gene, designated araG, contains an open reading frame capable of encoding an equally hydrophilic protein containing 504 residues (55,000 Mr). Following a 14-nucleotide spacer, which does not appear to have any secondary structure, the third open reading frame, herein designated araH, is capable of encoding a hydrophobic protein containing 329 residues (34,000 Mr) that can only be envisioned as having an integral membrane location. 3' to araH there is a T-rich region containing a 24-nucleotide area of dyad symmetry centered 55 nucleotides from the termination codon. Analysis of the derived primary sequences of the araG and araH products indicates the nature and potential features of these components. The araG protein was found to possess internal homology between its amino and carboxyl-terminal halves, suggesting a common origin. The araG gene product has been shown to be homologous to the rbsA gene product, the hisP product, the ptsB product and the malK product, all of which presumably play similar roles in their respective transport systems. Putative ATP binding sites are observed within the regions of homology. The araH gene product has been shown to be homologous to the rbsC gene product, which is the first observed homology between two purported membrane proteins.

  1. High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family*

    PubMed Central

    Dridi, Larbi; Ahmed Ouameur, Amin; Ouellette, Marc

    2010-01-01

    S-Adenosylmethionine (AdoMet) is an important methyl group donor that plays a central role in many essential biochemical processes. The parasite Leishmania can both synthesize and transport AdoMet. Leishmania cells resistant to the antifolate methotrexate due to a rearrangement in folate biopterin transporter (FBT) genes were cross-resistant to sinefungin, an AdoMet analogue. FBT gene rearrangements were also observed in Leishmania major cells selected for sinefungin resistance. One of the rearranged FBT genes corresponded to the main AdoMet transporter (AdoMetT1) of Leishmania as determined by gene transfection and gene inactivation experiments. AdoMetT1 was determined to be a high affinity plasma membrane transporter expressed constitutively throughout the growth phases of the parasite. Leishmania cells selected for resistance or naturally insensitive to sinefungin had lower expression of AdoMetT1. A new function in one carbon metabolism, also a pathway of interest for chemotherapeutic interventions, is described for a novel class of membrane proteins found in diverse organisms. PMID:20406813

  2. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    PubMed Central

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  3. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

    PubMed

    Ali, Shahin S; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose)) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

  4. TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.

    PubMed Central

    Gaber, R F; Styles, C A; Fink, G R

    1988-01-01

    We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion. Images PMID:3043197

  5. Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter.

    PubMed Central

    Kasahara, Toshiko; Kasahara, Michihiro

    2003-01-01

    Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator superfamily. Hxt1 shares approximately 70% amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports D-glucose with an affinity about one-tenth of that of Hxt2. To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt1 by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters. Among > 20000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced. With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8. We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8. Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2. The K (m) and V (max) values for D-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2. These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2. PMID:12603199

  6. Evolution of Plant Sucrose Uptake Transporters

    PubMed Central

    Reinders, Anke; Sivitz, Alicia B.; Ward, John M.

    2012-01-01

    In angiosperms, sucrose uptake transporters (SUTs) have important functions especially in vascular tissue. Here we explore the evolutionary origins of SUTs by analysis of angiosperm SUTs and homologous transporters in a vascular early land plant, Selaginella moellendorffii, and a non-vascular plant, the bryophyte Physcomitrella patens, the charophyte algae Chlorokybus atmosphyticus, several red algae and fission yeast, Schizosaccharomyces pombe. Plant SUTs cluster into three types by phylogenetic analysis. Previous studies using angiosperms had shown that types I and II are localized to the plasma membrane while type III SUTs are associated with vacuolar membrane. SUT homologs were not found in the chlorophyte algae Chlamydomonas reinhardtii and Volvox carterii. However, the characean algae Chlorokybus atmosphyticus contains a SUT homolog (CaSUT1) and phylogenetic analysis indicated that it is basal to all other streptophyte SUTs analyzed. SUTs are present in both red algae and S. pombe but they are less related to plant SUTs than CaSUT1. Both Selaginella and Physcomitrella encode type II and III SUTs suggesting that both plasma membrane and vacuolar sucrose transporter activities were present in early land plants. It is likely that SUT transporters are important for scavenging sucrose from the environment and intracellular compartments in charophyte and non-vascular plants. Type I SUTs were only found in eudicots and we conclude that they evolved from type III SUTs, possibly through loss of a vacuolar targeting sequence. Eudicots utilize type I SUTs for phloem (vascular tissue) loading while monocots use type II SUTs for phloem loading. We show that HvSUT1 from barley, a type II SUT, reverted the growth defect of the Arabidopsis atsuc2 (type I) mutant. This indicates that type I and II SUTs evolved similar (and interchangeable) phloem loading transporter capabilities independently. PMID:22639641

  7. A low K+ signal is required for functional high-affinity K+ uptake through HAK5 transporters.

    PubMed

    Rubio, Francisco; Fon, Mario; Ródenas, Reyes; Nieves-Cordones, Manuel; Alemán, Fernando; Rivero, Rosa M; Martínez, Vicente

    2014-11-01

    The high-affinity K(+) transporter HAK5 is a key system for root K(+) uptake and, under very low external K(+), the only one capable of supplying K(+) to the plant. Functional HAK5-mediated K(+) uptake should be tightly regulated for plant adaptation to different environmental conditions. Thus, it has been described that the gene encoding the transporter is transcriptionally regulated, being highly induced under K(+) limitation. Here we show that environmental conditions, such as the lack of K(+), NO(3)(-) or P, that induced a hyperpolarization of the plasma membrane of root cells, induce HAK5 transcription. However, only the deprivation of K(+) produces functional HAK5-mediated K(+) uptake in the root. These results suggest on the one hand the existence of a posttranscriptional regulation of HAK5 elicited by the low K(+) signal and on the other that HAK5 may be involved in yet-unknown functions related to NO(3)(-) and P deficiencies. These results have been obtained here with Solanum lycopersicum (cv. Micro-Tom) as well as Arabidopsis thaliana plants, suggesting that the posttranscriptional regulation of high-affinity HAK transporters take place in all plant species.

  8. Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

    PubMed

    Bagchi, Rammyani; Salehin, Mohammad; Adeyemo, O Sarah; Salazar, Carolina; Shulaev, Vladimir; Sherrier, D Janine; Dickstein, Rebecca

    2012-10-01

    The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.

  9. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  10. High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

    PubMed Central

    Nau-Wagner, Gabriele; Boch, Jens; Le Good, J. Ann; Bremer, Erhard

    1999-01-01

    We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a Ki of approximately 4 μM. Uptake studies with [1,2-dimethyl-14C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a Km value of 4 ± 1 μM and a maximum rate of transport (Vmax) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance 13C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline

  11. The fungal UmSrt1 and maize ZmSUT1 sucrose transporters battle for plant sugar resources.

    PubMed

    Wittek, Anke; Dreyer, Ingo; Al-Rasheid, Khaled A S; Sauer, Norbert; Hedrich, Rainer; Geiger, Dietmar

    2017-03-15

    The biotrophic fungus Ustilago maydis causes the corn smut disease inducing tumor formation in its host Zea mays. Upon infection, the fungal hyphae invaginate the plasma membrane of infected maize cells establishing an interface where pathogen and host are separated only by their plasma membranes. At this interface the fungal and maize sucrose transporters, UmSrt1 and ZmSUT1, compete for extracellular sucrose in the corn smut/maize pathosystem. Here we biophysically characterized ZmSUT1 and UmSrt1 in Xenopus oocytes with respect to their voltage-, pH- and substrate-dependence and determined affinities towards protons and sucrose. In contrast to ZmSUT1, UmSrt1 has a high-affinity for sucrose and is relatively pH- and voltage-independent. Using these quantitative parameters, we developed a mathematical model to simulate the competition for extracellular sucrose at the contact zone between the fungus and the host plant. This approach revealed that UmSrt1 exploits the apoplastic sucrose resource, which forces the plant transporter into a sucrose export mode providing the fungus with sugar from the phloem. Importantly, the high sucrose concentration in the phloem appeared disadvantageous for the ZmSUT1, preventing sucrose recovery from the apoplastic space in the fungus/plant interface.

  12. Regulation of the high-affinity copper transporter (hCtr1) expression by cisplatin and heavy metals.

    PubMed

    Liang, Zheng Dong; Long, Yan; Chen, Helen H W; Savaraj, Niramol; Kuo, Macus Tien

    2014-01-01

    Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.

  13. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    PubMed Central

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  14. Fructose Uptake in Sinorhizobium meliloti Is Mediated by a High-Affinity ATP-Binding Cassette Transport System

    PubMed Central

    Lambert, Annie; Østerås, Magne; Mandon, Karine; Poggi, Marie-Christine; Le Rudulier, Daniel

    2001-01-01

    By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (Km of 6 μM) fructose uptake system in S. meliloti. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of S. meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S. meliloti. The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is

  15. Fructose uptake in Sinorhizobium meliloti is mediated by a high-affinity ATP-binding cassette transport system.

    PubMed

    Lambert, A; Østerås, M; Mandon, K; Poggi, M C; Le Rudulier, D

    2001-08-01

    By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (K(m) of 6 microM) fructose uptake system in S. meliloti. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of S. meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S. meliloti. The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is

  16. Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

    PubMed

    Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles

    2008-03-28

    NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.

  17. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    PubMed

    Stockner, Thomas; Montgomery, Therese R; Kudlacek, Oliver; Weissensteiner, Rene; Ecker, Gerhard F; Freissmuth, Michael; Sitte, Harald H

    2013-01-01

    The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  18. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  19. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin.

    PubMed

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-03-20

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT.

  20. Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

    PubMed

    Cuddy, Leah K; Winick-Ng, Warren; Rylett, Rebecca Jane

    2014-03-01

    The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic

  1. Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model

    PubMed Central

    Stockner, Thomas; Montgomery, Therese R.; Kudlacek, Oliver; Weissensteiner, Rene; Ecker, Gerhard F.; Freissmuth, Michael; Sitte, Harald H.

    2013-01-01

    The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle. PMID:23436987

  2. Plant High-Affinity Potassium (HKT) Transporters involved in salinity tolerance: structural insights to probe differences in ion selectivity.

    PubMed

    Waters, Shane; Gilliham, Matthew; Hrmova, Maria

    2013-04-09

    High-affinity Potassium Transporters (HKTs) belong to an important class of integral membrane proteins (IMPs) that facilitate cation transport across the plasma membranes of plant cells. Some members of the HKT protein family have been shown to be critical for salinity tolerance in commercially important crop species, particularly in grains, through exclusion of Na+ ions from sensitive shoot tissues in plants. However, given the number of different HKT proteins expressed in plants, it is likely that different members of this protein family perform in a range of functions. Plant breeders and biotechnologists have attempted to manipulate HKT gene expression through genetic engineering and more conventional plant breeding methods to improve the salinity tolerance of commercially important crop plants. Successful manipulation of a biological trait is more likely to be effective after a thorough understanding of how the trait, genes and proteins are interconnected at the whole plant level. This article examines the current structural and functional knowledge relating to plant HKTs and how their structural features may explain their transport selectivity. We also highlight specific areas where new knowledge of plant HKT transporters is needed. Our goal is to present how knowledge of the structure of HKT proteins is helpful in understanding their function and how this understanding can be an invaluable experimental tool. As such, we assert that accurate structural information of plant IMPs will greatly inform functional studies and will lead to a deeper understanding of plant nutrition, signalling and stress tolerance, all of which represent factors that can be manipulated to improve agricultural productivity.

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

    PubMed

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

    2008-12-01

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

  4. Cloning, expression pattern and essentiality of the high-affinity copper transporter 1 (ctr1) gene in zebrafish.

    PubMed

    Mackenzie, Natalia C; Brito, Mónica; Reyes, Ariel E; Allende, Miguel L

    2004-03-17

    The high-affinity copper transporter 1 (Ctr1) is a highly conserved transmembrane protein that mediates the internalization of copper ions from the extracellular medium. In this study, we have isolated the zebrafish ctr1 gene. The zebrafish ctr1 cDNA encodes a protein with 69% identity to the human orthologue and shows conservation of specific amino acid residues involved in copper transport. We find only a single ctr1 gene in the zebrafish genome which maps to linkage group 5. The genomic structure of the zebrafish gene shows that it consists of five exons and that exon-intron boundaries are absolutely conserved with the mammalian ctr1 genes. Expression in embryos was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Zebrafish ctr1 is maternally loaded, and transcripts can be detected throughout development and in adult fish. Distribution of ctr1 message appears ubiquitous during early stages becoming restricted to the brain and ventral tissues by 24 h post fertilization (hpf). Beginning at 3 days post fertilization (dpf), expression is found mainly in the developing intestine. Specific knockdown of ctr1 by antisense morpholino oligonucleotides (MOs) causes early larval lethality. Defects include cell death in tissues where ctr1 is most heavily expressed, a finding similar to that described for a mouse knockout of mCtr1. Despite the existence of at least one other copper transport mechanism in the fish, our studies show that zebrafish ctr1 is an essential gene for development.

  5. A high affinity kidney targeting by chitobionic acid-conjugated polysorbitol gene transporter alleviates unilateral ureteral obstruction in rats.

    PubMed

    Islam, Mohammad Ariful; Kim, Sanghwa; Firdous, Jannatul; Lee, Ah-Young; Hong, Seong-Ho; Seo, Min Kyeong; Park, Tae-Eun; Yun, Cheol-Heui; Choi, Yun-Jaie; Chae, Chanhee; Cho, Chong-Su; Cho, Myung-Haing

    2016-09-01

    Aside from kidney transplantation - a procedure which is exceedingly dependent on donor-match and availability leading to excessive costs - there are currently no permanent treatments available which reverse kidney injury and failure. However, kidney-specific targeted gene therapy has outstanding potential to treat kidney-related dysfunction. Herein we report a novel kidney-specific targeted gene delivery system developed through the conjugation of chitobionic acid (CBA) to a polysorbitol gene transporter (PSGT) synthesized from sorbitol diacrylate and low molecular weight polyethylenimine (PEI) carrying hepatocyte growth factor (HGF) gene to alleviate unilateral ureteral obstruction (UUO) in rats. CBA-PSGT performed exceptionally well for targeted delivery of HGF to kidney tissues compared to its non-targeted counterparts (P < 0.001) after systemic tail-vein injection and significantly reduced the UUO symptoms, returning the UUO rats to a normal health status. The kidney-targeted CBA-PSGT-delivered HGF also strikingly reduced various pathologic and molecular markers in vivo such as the level of collagens (type I and II), blood urea nitrogen (BUN), creatinine, and the expressions of ICAM-1, TIMP-1 and α-SMA which play a critical role in obstructive kidney functions. Therefore, CBA-PSGT should be further investigated because of its potential to alleviate UUO and kidney-related diseases using high affinity kidney targeting.

  6. Characterization of Three Functional High-Affinity Ammonium Transporters in Lotus japonicus with Differential Transcriptional Regulation and Spatial Expression1

    PubMed Central

    D'Apuzzo, Enrica; Rogato, Alessandra; Simon-Rosin, Ulrike; El Alaoui, Hicham; Barbulova, Ani; Betti, Marco; Dimou, Maria; Katinakis, Panagiotis; Marquez, Antonio; Marini, Anne-Marie; Udvardi, Michael K.; Chiurazzi, Maurizio

    2004-01-01

    Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NH}}_{4}^{+}\\end{equation*}\\end{document} is also a regulator of early and late symbiotic interaction steps. Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation. Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus. Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant. Measurement of [14C]methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NH}}_{4}^{+}\\end{equation*}\\end{document}. The Ki for ammonium was 1.7, 3, and 15 μm for LjAMT1;1, 1;2, and 1;3, respectively. Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 > 1;2 > 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression. Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation. Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO2 concentrations, which also suppress photorespiration. Tissue and cellular localization of LjAMT1 genes expression, using promoter-β-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial

  7. Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1

    PubMed Central

    Gu, Chunsun; Song, Aiping; Zhang, Xiaoxue; Wang, Haibin; Li, Ting; Chen, Yu; Jiang, Jiafu; Chen, Fadi; Chen, Sumei

    2016-01-01

    The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter. PMID:27004464

  8. Platelets as potential peripheral markers to study functioning of the high-affinity sodium-dependent glutamate transporters in the nerve terminals of the brain

    NASA Astrophysics Data System (ADS)

    Borisova, T. A.; Kasatkina, L. A.

    Activity of the high-affinity sodium-dependent glutamate transporters in the brain nerve terminals is demonstrated to alter under artificial gravity conditions. A comparison analysis is made for L-[14C] glutamate transport in platelets and isolated nerve terminals. The kinetic characteristics of the transporters, [Na+]-dependence and influence of the transpoter inhibitor DL-threo-beta-benzyloxyaspartate on the L-[14C] glutamate uptake process are determined. It is shown that glutamate uptake process is very similar for platelets and nerve terminals. Thus it is reasonable to use platelets as a potential peripheral model for glutamate transport.

  9. The high-affinity metal Transporters NRAMP1 and IRT1 Team up to Take up Iron under Sufficient Metal Provision

    PubMed Central

    Castaings, Loren; Caquot, Antoine; Loubet, Stéphanie; Curie, Catherine

    2016-01-01

    Iron (Fe) and manganese (Mn) are essential metals which, when scarce in the growth medium, are respectively taken up by the root high affinity transporters IRT1 and NRAMP1 in Arabidopsis thaliana. The molecular bases for low affinity transport however remained unknown. Since IRT1 and NRAMP1 have a broad range of substrates among metals, we tested the hypothesis that they might be functionally redundant by generating nramp1 irt1 double mutants. These plants showed extreme Fe-deficiency symptoms despite optimal provision of the metal. Their phenotype, which includes low Fe and Mn contents and a defect of Fe entry into root cells as revealed by Fe staining, is rescued by high Fe supply. Using a promoter swap-based strategy, we showed that root endodermis retains the ability to carry out high affinity Fe transport and furthermore might be important to high-affinity Mn uptake. We concluded that NRAMP1 plays a pivotal role in Fe transport by cooperating with IRT1 to take up Fe in roots under replete conditions, thus providing the first evidence for a low affinity Fe uptake system in plants. PMID:27849020

  10. The high-affinity metal Transporters NRAMP1 and IRT1 Team up to Take up Iron under Sufficient Metal Provision.

    PubMed

    Castaings, Loren; Caquot, Antoine; Loubet, Stéphanie; Curie, Catherine

    2016-11-16

    Iron (Fe) and manganese (Mn) are essential metals which, when scarce in the growth medium, are respectively taken up by the root high affinity transporters IRT1 and NRAMP1 in Arabidopsis thaliana. The molecular bases for low affinity transport however remained unknown. Since IRT1 and NRAMP1 have a broad range of substrates among metals, we tested the hypothesis that they might be functionally redundant by generating nramp1 irt1 double mutants. These plants showed extreme Fe-deficiency symptoms despite optimal provision of the metal. Their phenotype, which includes low Fe and Mn contents and a defect of Fe entry into root cells as revealed by Fe staining, is rescued by high Fe supply. Using a promoter swap-based strategy, we showed that root endodermis retains the ability to carry out high affinity Fe transport and furthermore might be important to high-affinity Mn uptake. We concluded that NRAMP1 plays a pivotal role in Fe transport by cooperating with IRT1 to take up Fe in roots under replete conditions, thus providing the first evidence for a low affinity Fe uptake system in plants.

  11. Expression of high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae is both repressed and induced by glucose and appears to be regulated posttranslationally.

    PubMed Central

    Wendell, D L; Bisson, L F

    1994-01-01

    Expression of putative high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae was repressed 15- to 20-fold in high concentrations of glucose or fructose. S. cerevisiae with either the ssn6-delta 9 or the hxk2-delta 1::URA3 mutation, each of which relieves glucose repression, exhibited high Hxt2p expression in both 2.0% glucose (normally repressing) and 0.05% glucose (normally derepressing) while S. cerevisiae with the snf1-delta 10 mutation, which causes constitutive repression, did not detectably express Hxt2p in either glucose concentration. In addition to repressing at high concentrations, glucose or fructose is required for induction of Hxt2p expression. Hxt2p was not expressed by wild-type S. cerevisiae in media containing only ethanol or galactose as carbon and energy source but was expressed if glucose was added. An hxk2-delta 1::URA3 mutant did not detectably express Hxt2p in ethanol or galactose, but an ssn6-delta9 mutant did highly express Hxt2p in both carbon sources. Thus, simple relief of glucose repression as occurs with hxk2 null mutants is insufficient for high-level Hxt2p expression. Mutation of ssn6, a general transcriptional repressor, does lead to Hxt2p expression in the absence of glucose induction, suggesting relief of an additional negative regulatory system. High expression of Hxt2p does not always result in HXT2-dependent high-affinity transport, implying that Hxt2p activity is regulated posttranslationally. In the high glucose condition for the ssn6 mutant, high-affinity glucose transport is derepressed. Deletion of the HXT2 locus does not diminish this level of transport. However, high-affinity glucose transport is diminished in the ssn6-delta9 hxt2 delta1 double mutant compared with ssn6-delta9 alone in low glucose. Thus, while constitutively expressed in ssn6 mutants, Hxt2p only appears to be active as a transporter under low-glucose conditions. Similarly, Hxt2p was found to be expressed under low-glucose conditions

  12. The Organization of High-Affinity Ammonium Uptake in Arabidopsis Roots Depends on the Spatial Arrangement and Biochemical Properties of AMT1-Type Transporters[W

    PubMed Central

    Yuan, Lixing; Loqué, Dominique; Kojima, Soichi; Rauch, Sabine; Ishiyama, Keiki; Inoue, Eri; Takahashi, Hideki; von Wirén, Nicolaus

    2007-01-01

    The AMMONIUM TRANSPORTER (AMT) family comprises six isoforms in Arabidopsis thaliana. Here, we describe the complete functional organization of root-expressed AMTs for high-affinity ammonium uptake. High-affinity influx of 15N-labeled ammonium in two transposon-tagged amt1;2 lines was reduced by 18 to 26% compared with wild-type plants. Enrichment of the AMT1;2 protein in the plasma membrane and localization of AMT1;2 promoter activity in the endodermis and root cortex indicated that AMT1;2 mediates the uptake of ammonium entering the root via the apoplasmic transport route. An amt1;1 amt1;2 amt1;3 amt2;1 quadruple mutant (qko) showed severe growth depression under ammonium supply and maintained only 5 to 10% of wild-type high-affinity ammonium uptake capacity. Transcriptional upregulation of AMT1;5 in nitrogen-deficient rhizodermal and root hair cells and the ability of AMT1;5 to transport ammonium in yeast suggested that AMT1;5 accounts for the remaining uptake capacity in qko. Triple and quadruple amt insertion lines revealed in vivo ammonium substrate affinities of 50, 234, 61, and 4.5 μM for AMT1;1, AMT1;2, AMT1;3, and AMT1;5, respectively, but no ammonium influx activity for AMT2;1. These data suggest that two principle means of achieving effective ammonium uptake in Arabidopsis roots are the spatial arrangement of AMT1-type ammonium transporters and the distribution of their transport capacities at different substrate affinities. PMID:17693533

  13. Interleukin-2 and concanavalin A upregulate a cat2 isoform encoding a high affinity L-arginine transporter in feline lymphocytes.

    PubMed Central

    Stevens, B R; Tellier, M; Harvey, W; Feldman, D H; Bosworth, J

    2000-01-01

    The immunological responses of activated lymphocytes are associated with increased nitric oxide (NO) biosynthesis. Studies in the literature have primarily approached control of NO by focusing on the regulation of the nitric oxide synthase (NOS) isoforms. However, the present study approaches the control of NO synthesis by addressing the regulation of L-arginine availability to lymphocytes via regulation of membrane transport. The guanidino nitrogen of L-arginine is the sole biosynthetic precursor of NO. We investigated cytokine and mitogen regulation of membrane L-arginine transporters for the first time in feline cells. Feline peripheral blood mononuclear cells were treated with interleukin-2 and concanavalin A, then alternatively spliced isoforms of L-arginine transporters known in other species were probed by RT-PCR, using various oligonucleotide primers that hybridized to several regions in common with the isoforms. Both high affinity and low affinity isoforms are encoded by mRNAs arising from mutually exclusive alternative splicing of the primary transcript. A region of 123 bp was obtained that encoded an extracellular polypeptide loop of 41 amino acids. The sequence of this region represented the high affinity L-arginine substrate binding site of a CAT2 transporter polypeptide isoform, but not the CAT2a isoform low affinity binding site. Neither of the inducible isoforms were constitutively expressed in unstimulated feline cells. This is the first report demonstrating that domestic cats possess the cat2 gene encoding an inducible L-arginine transporter, and, furthermore, that the high affinity isoform transcript is activated by interleukin-2 and concanavalin A in feline lymphocytes. Images Figure 1. Figure 3. PMID:10935886

  14. Assignment of the gene coding for the human high-affinity glutamate transporter EAAC1 to 9p24: Potential role in dicarboxylic aminoaciduria and neurodegenerative disorders

    SciTech Connect

    Smith, C.P.; Kanai, Y.; Stelzner, M.; Hediger, M.A.; Weremowicz, S.; Morton, C.C. )

    1994-03-15

    Functional defects of high-affinity glutamate transporters have been implicated in the pathophysiology of neurodegenerative diseases such as amyotrophic lateral sclerosis. In small intestine and kidney, in which the high-affinity glutamate transporter mediates net absorption of glutamate and aspartate across epithelial cells, an inborn error of glutamate transport is thought to cause dicarboxylic aminoaciduria. This disorder is characterized by increased urinary excretion of glutamate and aspartate and is, in general, associated with neurologic and developmental abnormalities. Recently, the authors isolated a cDNA encoding a high-affinity glutamate transporter (EAAC1) that also transports aspartate but not other amino acids. EAAC1 is ubiquitously expressed throughout the body, particularly in brain (neurons), intestine, and kidney. Here, the authors present mapping of the chromosome location of EAAC1 using Southern analysis of a panel of human/rodent somatic cell hybrids and fluorescence in situ hybridization (FISH). Southern analysis of EcoRI-digested DNA gave bands at 6.5, 5.6, 5.1, and 1.2 kb for human genomic DNA; 7.5 kb for mouse genomic DNA; and 7.3, 3.2, and 1 kb for hamster genomic DNA. All four human EAAC1-specific bands were observed in the lane corresponding to the human/Chinese hamster hybrid containing chromosome 9 but not in lanes corresponding to any other hybrid. Because the human/Chinese hamster hybrid is the only one retaining chromosome 9, this result unambiguously assigns human EAAC1 to chromosome 9. For precise chromosome assignment of the human EAAC1 gene, they employed FISH. Map position of the EAAC1 probe was assigned by visual inspection of the fluorescent signal on the DAPI-stained metaphase chromosomes. The human EAAC1 gene was assigned to 9p24.

  15. High-Affinity Manganese Uptake by the Metal Transporter NRAMP1 Is Essential for Arabidopsis Growth in Low Manganese Conditions[C][W

    PubMed Central

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-01-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis. PMID:20228245

  16. Centrifuge-induced hypergravity and glutamate efflux by reversal of high-affinity, sodium-dependent transporters from rat brain synaptosomes.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Himmelreich, N.

    Glutamate uptake by high affinity sodium-dependent glutamate transporters is essential for termination of the synaptic transmission. Glutamate transporters may also contribute to an increase in extracellular glutamate. Glutamate efflux can occur by reversal of the sodium-dependent glutamate transporters during ATP depletion and dissipation of the sodium gradient across the cell membrane. Depolarization-induced calcium independent release of neurotransmitter from synaptosomal cytosolic pool is Na+-dependent and due to reverse of the neurotransmitter transporters also. We used monovalent organic cations N-methyl-D-glucamine (NMDG) to replace extracellular sodium, suggesting that the reducing of Na+ elucidate further the mechanism underlying Ca2+-independent glutamate release. A reduction in extracellular sodium would facilitate reversal of sodium-dependent transporters with extrusion of glutamate. We have compared the basal release of glutamate in Ca2+-free Na+-supplemented and NMDG-supplemented medium in control and after exposure of animals to long-arm centrifuge-induced hypergravity (ten G, during one hour). Replacement of sodium by NMDG enhanced basal level of neurotransmitter. The value of basal level increased to 110± 4% and 140± 2% in the medium with NMDG in comparison with Na+ under the control and hypergravity conditions, respectively. It is likely to reflect the enhancement of the neurotransmitter level in cytosolic pool. Thermodynamic considerations show that the extracellular level of a amino acid neurotransmitter, such as glutamate, that can be generated by transporter reversal are directly proportional to the intracellular concentration of the intracellular concentration of amino acid. KCl-stimulated glutamate release from cytosolic pool increased not statistically after hypergravity loading. We examined the effects of transporter inhibitors DL-threo-beta-benzyloxyaspartate ( DL-TBOA) on the release to elucidate whether reverse transport via the

  17. Crystallographic and spectroscopic evidence for high affinity binding of FeEDTA(H2O)- to the periplasmic nickel transporter NikA.

    PubMed

    Cherrier, Mickaël V; Martin, Lydie; Cavazza, Christine; Jacquamet, Lilian; Lemaire, David; Gaillard, Jacques; Fontecilla-Camps, Juan C

    2005-07-20

    Because nickel is both essential and toxic to a great variety of organisms, its detection and transport is highly regulated. In Escherichia coli and other related Gram-negative bacteria, high affinity nickel transport depends on proteins expressed by the nik operon. A central actor of this process is the periplasmic NikA transport protein. A previous structural report has proposed that nickel binds to NikA as a pentahydrate species. However, both stereochemical considerations and X-ray absorption spectroscopic results are incompatible with that interpretation. Here, we report the 1.8 A resolution structure of NikA and show that it binds FeEDTA(H2O)- with very high affinity. In addition, we provide crystallographic evidence that a metal-EDTA complex was also bound to the previously reported NikA structure. Our observations strongly suggest that nickel transport in E. coli requires the binding of this metal ion to a metallophore that bears significant resemblance to EDTA. They also provide a basis for the potential use of NikA in the bioremediation of toxic transition metals and the design of artificial metalloenzymes.

  18. Pharmacological characterization of N,N-dimethyl-2-(2-amino-4-methylphenyl thio)benzylamine as a ligand of the serotonin transporter with high affinity and selectivity.

    PubMed

    Chalon, Sylvie; Tarkiainen, Jari; Garreau, Lucette; Hall, Hakan; Emond, Patrick; Vercouillie, Johnny; Farde, Lars; Dasse, Philippe; Varnas, Katarina; Besnard, Jean-Claude; Halldin, Christer; Guilloteau, Denis

    2003-01-01

    Serotonin transporter has a key-role in regulation of serotoninergic function, and is involved in numerous neurodegenerative and psychiatric disorders. To obtain an efficient radioactive ligand allowing the study of this transporter in vitro and in vivo, we synthesized a new diphenyl sulfide derivative, N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine or MADAM. We present here extensive pharmacological characterization of this compound. [3H]MADAM bound to serotonin transporters with a very high affinity in vitro on rat cortical membranes, at least 2 times better than the most commonly used radioactive probes (Kd, 60 pM; Bmax, 543 fmol/mg of protein). Competition studies showed few inhibitory effect of nisoxetine (Ki = 270 nM), no inhibitory effect of desipramine or 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935) (Ki >1000 nM), and strong effect of paroxetine (Ki = 0.32 nM) and citalopram (Ki = 1.57 nM). Therefore, MADAM has around 1000-fold better selectivity for the serotonin transporter than for other transporters. Autoradiographic studies both on rat and postmortem human brain slices demonstrated that the distribution of [3H]MADAM parallels the localization of serotonin transporters and is prevented by known inhibitors of them. The high affinity and selectivity of [3H]MADAM for the serotonin transporter show that it is very valuable for studies using in vitro approaches. The high selectivity and low nonspecific binding of [3H]MADAM on the postmortem human brain, together with preliminary in vivo results with [11C]MADAM, is a new argument for future use of this ligand in in vivo studies of the distribution, pharmacology, and pathophysiology of the serotonin transporter in the human brain with positron emission tomography.

  19. The sea urchin embryo as a model for mammalian developmental neurotoxicity: ontogenesis of the high-affinity choline transporter and its role in cholinergic trophic activity.

    PubMed

    Qiao, Dan; Nikitina, Lyudmila A; Buznikov, Gennady A; Lauder, Jean M; Seidler, Frederic J; Slotkin, Theodore A

    2003-11-01

    Embryonic development in the sea urchin requires trophic actions of the same neurotransmitters that participate in mammalian brain assembly. We evaluated the development of the high-affinity choline transporter, which controls acetylcholine synthesis. A variety of developmental neurotoxicants affect this transporter in mammalian brain. [3H]Hemicholinium-3 binding to the transporter was found in the cell membrane fraction at stages from the unfertilized egg to pluteus, with a binding affinity comparable with that seen in mammalian brain. Over the course of development, the concentration of transporter sites rose more than 3-fold, achieving concentrations comparable with those of cholinergically enriched mammalian brain regions. Dimethylaminoethanol (DMAE), a competitive inhibitor of choline transport, elicited dysmorphology beginning at the mid-blastula stage, with anomalies beginning progressively later as the concentration of DMAE was lowered. Pretreatment, cotreatment, or delayed treatment with acetylcholine or choline prevented the adverse effects of DMAE. Because acetylcholine was protective at a lower threshold, the DMAE-induced defects were most likely mediated by its effects on acetylcholine synthesis. Transient removal of the hyaline layer enabled a charged transport inhibitor, hemicholinium-3, to penetrate sufficiently to elicit similar anomalies, which were again prevented by acetylcholine or choline. These results indicate that the developing sea urchin possesses a high-affinity choline transporter analogous to that found in the mammalian brain, and, as in mammals, the functioning of this transporter plays a key role in the developmental, trophic activity of acetylcholine. The sea urchin model may thus be useful in high-throughput screening of suspected developmental neurotoxicants.

  20. Sucrose transporters in two members of the Scrophulariaceae with different types of transport sugar.

    PubMed

    Knop, C; Voitsekhovskaja, O; Lohaus, G

    2001-05-01

    In order to study differences between sugar transport in oligosaccharide-translocating and sucrose-translocating species, two members of the Scrophulariaceae, Asarina barclaiana Pennell and Alonsoa meridionalis O. Kuntze, were analysed regarding minor-vein anatomy, sugar concentrations in leaves and phloem sap, and expression of sucrose transporters. The minor veins of Asarina barclaiana possess mainly transfer cells and modified intermediary cells and those of Alonsoa meridionalis have intermediary cells and ordinary companion cells. Phloem sap from these plants was collected by the laser-aphid-stylet technique. The main carbon transport forms in Asarina were sucrose and in Alonsoa raffinose and stachyose. The sum of the carbohydrate concentrations in the phloem sap of both species was as high as that in apoplastic phloem loaders. In Asarina the ratio of the sucrose concentration in the phloem to that in the cytosol of source cells was about 35 and the corresponding ratio in Alonsoa was about two. Sucrose transporter cDNAs were isolated from leaves of both species. By means of semi-quantitative reverse transcription-polymerase chain reaction, sucrose transporter mRNA was detected in different organs and also in the phloem sap. This is the first time that sucrose transporters have been found in oligosaccharide-translocating species and that the mRNA of these sucrose transporters has been localized directly in the phloem sap. Taken together, our observations indicate that Asarina is an apoplastic phloem loader, while the results for Alonsoa are ambiguous: some properties are typical of the symplastic phloem-loading mechanism, but probably a sucrose transporter is involved in loading and/or retrieval of sucrose into the phloem.

  1. Induction of the high-affinity Na(+)-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1.

    PubMed Central

    Ferrer-Martinez, A; Felipe, A; Nicholson, B; Casado, J; Pastor-Anglada, M; McGivan, J

    1995-01-01

    The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself. Images Figure 4 PMID:7654212

  2. Capacity and Plasticity of Potassium Channels and High-Affinity Transporters in Roots of Barley and Arabidopsis1[C][W

    PubMed Central

    Coskun, Devrim; Britto, Dev T.; Li, Mingyuan; Oh, Saehong; Kronzucker, Herbert J.

    2013-01-01

    The role of potassium (K+) transporters in high- and low-affinity K+ uptake was examined in roots of intact barley (Hordeum vulgare) and Arabidopsis (Arabidopsis thaliana) plants by use of 42K radiotracing, electrophysiology, pharmacology, and mutant analysis. Comparisons were made between results from barley and five genotypes of Arabidopsis, including single and double knockout mutants for the high-affinity transporter, AtHAK5, and the Shaker-type channel, AtAKT1. In Arabidopsis, steady-state K+ influx at low external K+ concentration ([K+]ext = 22.5 µm) was predominantly mediated by AtAKT1 when high-affinity transport was inhibited by ammonium, whereas in barley, by contrast, K+ channels could not operate below 100 µm. Withdrawal of ammonium resulted in an immediate and dramatic stimulation of K+ influx in barley, indicating a shift from active to passive K+ uptake at low [K+]ext and yielding fluxes as high as 36 µmol g (root fresh weight)−1 h−1 at 5 mm [K+]ext, among the highest transporter-mediated K+ fluxes hitherto reported. This ammonium-withdrawal effect was also established in all Arabidopsis lines (the wild types, atakt1, athak5, and athak5 atakt1) at low [K+]ext, revealing the concerted involvement of several transport systems. The ammonium-withdrawal effect coincided with a suppression of K+ efflux and a significant hyperpolarization of the plasma membrane in all genotypes except athak5 atakt1, could be sustained over 24 h, and resulted in increased tissue K+ accumulation. We discuss key differences and similarities in K+ acquisition between two important model systems and reveal novel aspects of K+ transport in planta. PMID:23553635

  3. Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

    SciTech Connect

    Zhang, Xinxin; Zhang, Min; Takano, Tetsuo; Liu, Shenkui

    2011-10-14

    Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.

  4. Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice

    PubMed Central

    Nguyen, Hai T; Guiard, Bruno P; Bacq, Alexandre; David, Denis J; David, Indira; Quesseveur, Gaël; Gautron, Sophie; Sanchez, Connie; Gardier, Alain M

    2013-01-01

    BACKGROUND AND PURPOSE Escitalopram, the S(+)-enantiomer of citalopram is the most selective 5-HT reuptake inhibitor approved. Although all 5-HT selective reuptake inhibitors (SSRIs) increase extracellular levels of 5-HT ([5-HT]ext). some also enhance, to a lesser extent, extracellular levels of noradrenaline ([NA]ext). However, the mechanisms by which SSRIs activate noradrenergic transmission in the brain remain to be determined. EXPERIMENTAL APPROACH This study examined the effects of escitalopram, on both [5-HT]ext and [NA]ext in the frontal cortex (FCx) of freely moving wild-type (WT) and mutant mice lacking the 5-HT transporter (SERT−/−) by using intracerebral microdialysis. We explored the possibilities that escitalopram enhances [NA]ext, either by a direct mechanism involving the inhibition of the low- or high-affinity noradrenaline transporters, or by an indirect mechanism promoted by [5-HT]ext elevation. The forced swim test (FST) was used to investigate whether enhancing cortical [5-HT]ext and/or [NA]ext affected the antidepressant-like activity of escitalopram. KEY RESULTS In WT mice, a single systemic administration of escitalopram produced a significant increase in cortical [5-HT]ext and [NA]ext. As expected, escitalopram failed to increase cortical [5-HT]ext in SERT−/− mice, whereas its neurochemical effects on [NA]ext persisted in these mutants. In WT mice subjected to the FST, escitalopram increased swimming parameters without affecting climbing behaviour. Finally, escitalopram, at relevant concentrations, failed to inhibit cortical noradrenaline and 5-HT uptake mediated by low-affinity monoamine transporters. CONCLUSIONS AND IMPLICATIONS These experiments suggest that escitalopram enhances, although moderately, cortical [NA]extin vivo by a direct mechanism involving the inhibition of the high-affinity noradrenaline transporter (NET). PMID:22233336

  5. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

  6. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis

    PubMed Central

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-01-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose–H+ symporter. Analyses of STP10 mRNA and STP10 promoter–reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10. qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10 T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway. PMID:26893494

  7. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis.

    PubMed

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-04-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose-H(+)symporter. Analyses of STP10 mRNA and STP10 promoter-reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10 qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway.

  8. Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells.

    PubMed

    Chan, Helen; Zwingmann, Claudia; Pannunzio, Marc; Butterworth, Roger F

    2003-07-01

    Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on D-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM for 5 days, P<0.01) suppression of D-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake (V(max)) (37% decrease at 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at

  9. [Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

    PubMed

    Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

    2012-01-01

    It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

  10. Components of the Vid30c are needed for the rapamycin-induced degradation of the high-affinity hexose transporter Hxt7p in Saccharomyces cerevisiae.

    PubMed

    Snowdon, Chris; Hlynialuk, Chris; van der Merwe, George

    2008-03-01

    Saccharomyces cerevisiae adapts to changing nutrient conditions by regulating its genome-wide transcription profile and cell-wide protein complement in correlation with the reigning nutrient conditions. The target of rapamycin (TOR) signalling pathway is one of the major control mechanisms within the cell that facilitates these changes. The transcription, intracellular trafficking, and protein turnover of nutrient transporters, including the hexose transporter proteins (Hxts), are regulated in response to nutrient conditions. The Vid and Gid proteins facilitate the nutrient-dependent degradation of the gluconeogenic enzymes FBPase and Mdh2p when glucose-starved cells are replenished with glucose. Three members of the VID and GID gene families, VID30/GID1, GID2, and VID28/GID5 are needed for the rapamycin or nitrogen starvation-induced degradation of the high-affinity hexose transporter Hxt7p is shown here. In addition, evidence that the functions of several Vid and Gid proteins are in close relation to the TOR signalling pathway is provided.

  11. Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2

    PubMed Central

    He, Xiao; Garza, Denisse; Nigam, Sanjay K.; Chang, Geoffrey

    2016-01-01

    Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates—in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1—prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with KD values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (ka) in the range of 103 M-1s-1 and an even slower dissociation rate (kd) in the range of 10−4 s-1 for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1—which is normally expressed in liver and other tissues—interacts with prostaglandin analogs. While it is not

  12. Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2.

    PubMed

    He, Xiao; Garza, Denisse; Nigam, Sanjay K; Chang, Geoffrey

    2016-01-01

    Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates--in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1-prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with KD values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (ka) in the range of 103 M(-1) s(-1) and an even slower dissociation rate (kd) in the range of 10-4 s(-1) for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1--which is normally expressed in liver and other tissues--interacts with prostaglandin analogs. While it is not

  13. Aspen SUCROSE TRANSPORTER3 allocates carbon into wood fibers.

    PubMed

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

    2013-12-01

    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 (13)CO2 assimilation, the SUT3RNAi lines contained more (13)C 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.

  14. Cardiovascular afferents cause the release of 5-HT in the nucleus tractus solitarii; this release is regulated by the low- (PMAT) not the high-affinity transporter (SERT)

    PubMed Central

    Hosford, Patrick S; Millar, Julian; Ramage, Andrew G

    2015-01-01

    Key points The nucleus tractus solitarii (NTS) integrates visceral afferent information essential for cardiovascular haemostasis. Using fast-cyclic voltammetry in anaesthetized rats, 5-HT (serotonin) release was detected in NTS in response to activation of these afferents. Removal of 5-HT from the extracellular space is usually regulated by the low-capacity, high-affinity 5-HT transporter (5-HTT/SERT). The present data demonstrate that 5-HT removal in the NTS is regulated by the plasma membrane monoamine transporter (PMAT), a high-capacity, low-affinity transporter. The present data also demonstrate that the 5-HT released by afferent activation comes from at least two different sources. It is suggested that one of these sources is the afferents themselves. These results demonstrate a physiological role for the low-affinity uptake transporter in the regulation of 5-HT concentration in NTS. Abstract The nucleus tractus solitarii (NTS) integrates inputs from cardiovascular afferents and thus is crucial for cardiovascular homeostasis. These afferents primarily release glutamate, although 5-HT has also been shown to play a role in their actions. Using fast-cyclic voltammetry, an increase in 5-HT concentrations (range 12–50 nm) could be detected in the NTS in anaesthetized rats in response to electrical stimulation of the vagus and activation of cardiopulmonary, chemo- and baroreceptor reflexes. This 5-HT signal was not potentiated by the serotonin transporter (SERT) or the noradrenaline transporter (NET) inhibitors citalopram and desipramine (1 mg kg−1). However, decynium-22 (600 μg kg−1), an organic cation 3 transporter (OCT3)/plasma membrane monoamine transporter (PMAT) inhibitor, increased the 5-HT signal by 111 ± 21% from 29 ± 10 nm. The effectiveness of these inhibitors was tested against the removal time of 5-HT and noradrenaline applied by microinjection to the NTS. Citalopram and decynium-22 attenuated the removal of 5-HT but not

  15. Deletion of SLC19A2, the high affinity thiamine transporter, causes selective inner hair cell loss and an auditory neuropathy phenotype.

    PubMed

    Liberman, M C; Tartaglini, E; Fleming, J C; Neufeld, E J

    2006-09-01

    Mutations in the gene coding for the high-affinity thiamine transporter Slc19a2 underlie the clinical syndrome known as thiamine-responsive megaloblastic anemia (TRMA) characterized by anemia, diabetes, and sensorineural hearing loss. To create a mouse model of this disease, a mutant line was created with targeted disruption of the gene. Cochlear function is normal in these mutants when maintained on a high-thiamine diet. When challenged with a low-thiamine diet, Slc19a2-null mice showed 40-60 dB threshold elevations by auditory brainstem response (ABR), but only 10-20 dB elevation by otoacoustic emission (OAE) measures. Wild-type mice retain normal hearing on either diet. Cochlear histological analysis showed a pattern uncommon for sensorineural hearing loss: selective loss of inner hair cells after 1-2 weeks on low thiamine and significantly greater inner than outer hair cell loss after longer low-thiamine challenges. Such a pattern is consistent with the observed discrepancy between ABR and OAE threshold shifts. The possible role of thiamine transport in other reported cases of selective inner hair cell loss is considered.

  16. Knockdown of the partner protein OsNAR2.1 for high-affinity nitrate transport represses lateral root formation in a nitrate-dependent manner.

    PubMed

    Huang, Shuangjie; Chen, Si; Liang, Zhihao; Zhang, Chenming; Yan, Ming; Chen, Jingguang; Xu, Guohua; Fan, Xiaorong; Zhang, Yali

    2015-12-08

    The morphological plasticity of root systems is critical for plant survival, and understanding the mechanisms underlying root adaptation to nitrogen (N) fluctuation is critical for sustainable agriculture; however, the molecular mechanism of N-dependent root growth in rice remains unclear. This study aimed to identify the role of the complementary high-affinity NO3(-) transport protein OsNAR2.1 in NO3(-)-regulated rice root growth. Comparisons with wild-type (WT) plants showed that knockdown of OsNAR2.1 inhibited lateral root (LR) formation under low NO3(-) concentrations, but not under low NH4(+) concentrations. (15)N-labelling NO3(-) supplies (provided at concentrations of 0-10 mM) demonstrated that (i) defects in LR formation in mutants subjected to low external NO3(-) concentrations resulted from impaired NO3(-) uptake, and (ii) the mutants had significantly fewer LRs than the WT plants when root N contents were similar between genotypes. LR formation in osnar2.1 mutants was less sensitive to localised NO3(-) supply than LR formation in WT plants, suggesting that OsNAR2.1 may be involved in a NO3(-)-signalling pathway that controls LR formation. Knockdown of OsNAR2.1 inhibited LR formation by decreasing auxin transport from shoots to roots. Thus, OsNAR2.1 probably functions in both NO3(-) uptake and NO3(-)-signalling.

  17. The Rice High-Affinity Potassium Transporter1;1 Is Involved in Salt Tolerance and Regulated by an MYB-Type Transcription Factor.

    PubMed

    Wang, Rong; Jing, Wen; Xiao, Longyun; Jin, Yakang; Shen, Like; Zhang, Wenhua

    2015-07-01

    Sodium transporters play key roles in plant tolerance to salt stress. Here, we report that a member of the High-Affinity K(+) Transporter (HKT) family, OsHKT1;1, in rice (Oryza sativa 'Nipponbare') plays an important role in reducing Na(+) accumulation in shoots to cope with salt stress. The oshkt1;1 mutant plants displayed hypersensitivity to salt stress. They contained less Na(+) in the phloem sap and accumulated more Na(+) in the shoots compared with the wild type. OsHKT1;1 was expressed mainly in the phloem of leaf blades and up-regulated in response to salt stress. Using a yeast one-hybrid approach, a novel MYB coiled-coil type transcription factor, OsMYBc, was found to bind to the OsHKT1;1 promoter. In vivo chromatin immunoprecipitation and in vitro electrophoresis mobility shift assays demonstrated that OsMYBc binds to AAANATNC(C/T) fragments within the OsHKT1;1 promoter. Mutation of the OsMYBc-binding nucleotides resulted in a decrease in promoter activity of OsHKT1;1. Knockout of OsMYBc resulted in a reduction in NaCl-induced expression of OsHKT1;1 and salt sensitivity. Taken together, these results suggest that OsHKT1;1 has a role in controlling Na(+) concentration and preventing sodium toxicity in leaf blades and is regulated by the OsMYBc transcription factor.

  18. Aspects of sucrose transport in stem parenchyma of sweet sorghum. [Sorghum bicolor

    SciTech Connect

    Lingle, S.E.

    1987-08-01

    Sweet sorghum (Sorghum bicolor (L.) Moench) is a sucrose-storing crop with a storage tissue anatomically similar to that of sugarcane (Saccharum spp.). However, recent evidence suggests that sweet sorghum may be biochemically different from sugarcane. /sup 14/C-sucrose uptake was studied in excised tissue discs from fully-elongated internodes of Rio sweet sorghum. Washout studies gave results consistent with a 3 compartment system. After 3 hours of uptake, most of the /sup 14/C was found in the vacuole compartment, and was determined by HPLC to be sucrose. Total sucrose uptake consisted of a PCMBS-sensitive (active) and a PCMBS-insensitive (passive) component. Active sucrose uptake had a pH optimum of 4.5. Total sucrose uptake was negatively correlated with the internal sucrose content of the tissue. Fructosyl-labelled /sup 14/C-sucrose was not randomized during uptake, suggesting that sucrose cleavage is not a requirement for sucrose uptake in sweet sorghum. This data suggests that in sweet sorghum, sucrose is transported intact by a specific carrier, as opposed to the sucrose-cleavage-and-resynthesis transport system that apparently operates in sugarcane.

  19. Tonoplast Sugar Transporters (SbTSTs) putatively control sucrose accumulation in sweet sorghum stems.

    PubMed

    Bihmidine, Saadia; Julius, Benjamin T; Dweikat, Ismail; Braun, David M

    2016-01-01

    Carbohydrates are differentially partitioned in sweet versus grain sorghums. While the latter preferentially accumulate starch in the grain, the former primarily store large amounts of sucrose in the stem. Previous work determined that neither sucrose metabolizing enzymes nor changes in Sucrose transporter (SUT) gene expression accounted for the carbohydrate partitioning differences. Recently, 2 additional classes of sucrose transport proteins, Tonoplast Sugar Transporters (TSTs) and SWEETs, were identified; thus, we examined whether their expression tracked sucrose accumulation in sweet sorghum stems. We determined 2 TSTs were differentially expressed in sweet vs. grain sorghum stems, likely underlying the massive difference in sucrose accumulation. A model illustrating potential roles for different classes of sugar transport proteins in sorghum sugar partitioning is discussed.

  20. Tonoplast Sugar Transporters (SbTSTs) putatively control sucrose accumulation in sweet sorghum stems

    PubMed Central

    Bihmidine, Saadia; Julius, Benjamin T; Dweikat, Ismail; Braun, David M

    2016-01-01

    ABSTRACT Carbohydrates are differentially partitioned in sweet versus grain sorghums. While the latter preferentially accumulate starch in the grain, the former primarily store large amounts of sucrose in the stem. Previous work determined that neither sucrose metabolizing enzymes nor changes in Sucrose transporter (SUT) gene expression accounted for the carbohydrate partitioning differences. Recently, 2 additional classes of sucrose transport proteins, Tonoplast Sugar Transporters (TSTs) and SWEETs, were identified; thus, we examined whether their expression tracked sucrose accumulation in sweet sorghum stems. We determined 2 TSTs were differentially expressed in sweet vs. grain sorghum stems, likely underlying the massive difference in sucrose accumulation. A model illustrating potential roles for different classes of sugar transport proteins in sorghum sugar partitioning is discussed. PMID:26619184

  1. Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1.

    PubMed

    Li, Guowei; Tillard, Pascal; Gojon, Alain; Maurel, Christophe

    2016-04-01

    The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3(-)), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To explore the effects and mode of action of NO3(-) on Lpr, we used an extended set of NO3(-) transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1 and nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3(-) conditions. First, a strong positive relationship between Lpr and NO3(-) accumulation, in shoots rather than in roots, was revealed. Secondly, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role for the high-affinity NO3(-) transporter NRT2.1 in increasing Lpr These results indicate that NO3(-)signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative real-time reverse transcription-PCR and enzyme-linked immunosorbent assays (ELISAs) were used to investigate the effects of NO3(-) availability on plasma membrane aquaporin (plasma membrane intrinsic protein; PIP) expression. Whereas PIP regulation mostly occurs at the post-translational level in wild-type plants, a regulation of PIPs at both the transcriptional and translational levels was uncovered in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3(-) involves novel shoot to root signaling and NRT2.1-dependent functions.

  2. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    PubMed Central

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-01-01

    AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants. PMID:25915023

  3. Unequal functional redundancy between the two Arabidopsis thaliana high-affinity sulphate transporters SULTR1;1 and SULTR1;2.

    PubMed

    Barberon, Marie; Berthomieu, Pierre; Clairotte, Michael; Shibagaki, Nakako; Davidian, Jean-Claude; Gosti, Françoise

    2008-01-01

    * In Arabidopsis, SULTR1;1 and SULTR1;2 are two genes proposed to be involved in high-affinity sulphate uptake from the soil solution. We address here the specific issue of their functional redundancy for the uptake of sulphate and for the accumulation of its toxic analogue selenate with regard to plant growth and selenate tolerance. * Using the complete set of genotypes, including the wild-type, each one of the single sultr1;1 and sultr1;2 mutants and the resulting double sultr1;1-sultr1;2 mutant, we performed a detailed phenotypic analysis of root length, shoot biomass, sulphate uptake, sulphate and selenate accumulation and selenate tolerance. * The results all ordered the four different genotypes according to the same functional hierarchy. Wild-type and sultr1;1 mutant plants displayed similar phenotypes. By contrast, sultr1;1-sultr1;2 double-mutant plants showed the most extreme phenotype and the sultr1;2 mutant displayed intermediate performances. Additionally, the degree of selenate tolerance was directly related to the seedling selenate content according to a single sigmoid regression curve common to all the genotypes. * The SULTR1;1 and SULTR1;2 genes display unequal functional redundancy, which leaves open for SULTR1;1 the possibility of displaying an additional function besides its role in sulphate membrane transport.

  4. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status.

    PubMed

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-04-23

    AMT1-3 encodes the high affinity NH₄⁺ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

  5. Differential regulation of two sucrose transporters by defoliation and light conditions in perennial ryegrass.

    PubMed

    Furet, Pierre-Maxime; Berthier, Alexandre; Decau, Marie-Laure; Morvan-Bertrand, Annette; Prud'homme, Marie-Pascale; Noiraud-Romy, Nathalie; Meuriot, Frédéric

    2012-12-01

    Sucrose transport between source and sink tissues is supposed to be a key-step for an efficient regrowth of perennial rye-grass after defoliation and might be altered by light conditions. We assessed the effect of different light regimes (high vs low light applied before or after defoliation) on growth, fructans and sucrose mobilization, as well as on sucrose transporter expression during 14 days of regrowth. Our results reported that defoliation led to a mobilization of C reserves (first sucrose and then fructans), which was parallel to an induction of LpSUT1 sucrose transporter expression in source and sink tissues (i.e. leaf sheaths and elongating leaf bases, respectively) irrespective to light conditions. Light regime (high or low light) had little effects on regrowth and on C reserves mobilization during the first 48 h of regrowth after defoliation. Thereafter, low light conditions, delaying the recovery of photosynthetic capacities, had a negative effect on C reserves re-accumulation (especially sucrose). Surprisingly, high light did not enhance sucrose transporter expression. Indeed, while light conditions had no effect on LpSUT1 expression, LpSUT2 transcripts levels were enhanced for low light grown plants. These results indicate that two sucrose transporter currently identified in Lolium perenne L. are differentially regulated by light and sucrose.

  6. Knockdown of the partner protein OsNAR2.1 for high-affinity nitrate transport represses lateral root formation in a nitrate-dependent manner

    PubMed Central

    Huang, Shuangjie; Chen, Si; Liang, Zhihao; Zhang, Chenming; Yan, Ming; Chen, Jingguang; Xu, Guohua; Fan, Xiaorong; Zhang, Yali

    2015-01-01

    The morphological plasticity of root systems is critical for plant survival, and understanding the mechanisms underlying root adaptation to nitrogen (N) fluctuation is critical for sustainable agriculture; however, the molecular mechanism of N-dependent root growth in rice remains unclear. This study aimed to identify the role of the complementary high-affinity NO3− transport protein OsNAR2.1 in NO3−-regulated rice root growth. Comparisons with wild-type (WT) plants showed that knockdown of OsNAR2.1 inhibited lateral root (LR) formation under low NO3− concentrations, but not under low NH4+ concentrations. 15N-labelling NO3− supplies (provided at concentrations of 0–10 mM) demonstrated that (i) defects in LR formation in mutants subjected to low external NO3− concentrations resulted from impaired NO3− uptake, and (ii) the mutants had significantly fewer LRs than the WT plants when root N contents were similar between genotypes. LR formation in osnar2.1 mutants was less sensitive to localised NO3− supply than LR formation in WT plants, suggesting that OsNAR2.1 may be involved in a NO3−-signalling pathway that controls LR formation. Knockdown of OsNAR2.1 inhibited LR formation by decreasing auxin transport from shoots to roots. Thus, OsNAR2.1 probably functions in both NO3− uptake and NO3−-signalling. PMID:26644084

  7. Structures of Streptococcus pneumoniae PiaA and Its Complex with Ferrichrome Reveal Insights into the Substrate Binding and Release of High Affinity Iron Transporters

    PubMed Central

    Cheng, Wang; Li, Qiong; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing

    2013-01-01

    Iron scarcity is one of the nutrition limitations that the Gram-positive infectious pathogens Streptococcus pneumoniae encounter in the human host. To guarantee sufficient iron supply, the ATP binding cassette (ABC) transporter Pia is employed to uptake iron chelated by hydroxamate siderophore, via the membrane-anchored substrate-binding protein PiaA. The high affinity towards ferrichrome enables PiaA to capture iron at a very low concentration in the host. We presented here the crystal structures of PiaA in both apo and ferrichrome-complexed forms at 2.7 and 2.1 Å resolution, respectively. Similar to other class III substrate binding proteins, PiaA is composed of an N-terminal and a C-terminal domain bridged by an α-helix. At the inter-domain cleft, a molecule of ferrichrome is stabilized by a number of highly conserved residues. Upon ferrichrome binding, two highly flexible segments at the entrance of the cleft undergo significant conformational changes, indicating their contribution to the binding and/or release of ferrichrome. Superposition to the structure of Escherichia coli ABC transporter BtuF enabled us to define two conserved residues: Glu119 and Glu262, which were proposed to form salt bridges with two arginines of the permease subunits. Further structure-based sequence alignment revealed that the ferrichrome binding pattern is highly conserved in a series of PiaA homologs encoded by both Gram-positive and negative bacteria, which were predicted to be sensitive to albomycin, a sideromycin antibiotic derived from ferrichrome. PMID:23951167

  8. Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation.

    PubMed

    Boscari, Alexandre; Van de Sype, Ghislaine; Le Rudulier, Daniel; Mandon, Karine

    2006-08-01

    Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (beta-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the overexpressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

  9. LjMOT1, a high-affinity molybdate transporter from Lotus japonicus, is essential for molybdate uptake, but not for the delivery to nodules.

    PubMed

    Duan, Guilan; Hakoyama, Tsuneo; Kamiya, Takehiro; Miwa, Hiroki; Lombardo, Fabien; Sato, Shusei; Tabata, Satoshi; Chen, Zheng; Watanabe, Toshihiro; Shinano, Takuro; Fujiwara, Toru

    2017-03-09

    Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high-affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate-mutagenized line (ljmot1) decreased by 70-95% compared with wild type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp(292) to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F2 progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60-70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the Km was 182 nM. LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L. japonicus for Mo uptake from the soil and growth, but is not Mo delivery to the nodules. This article is protected by copyright. All rights reserved.

  10. Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms.

    PubMed

    Santi, Simonetta; Locci, Geraldine; Monte, Rossella; Pinton, Roberto; Varanini, Zeno

    2003-08-01

    An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.

  11. Time-Resolved Investigation of Molecular Components Involved in the Induction of [Formula: see text] High Affinity Transport System in Maize Roots.

    PubMed

    Pii, Youry; Alessandrini, Massimiliano; Dall'Osto, Luca; Guardini, Katia; Prinsi, Bhakti; Espen, Luca; Zamboni, Anita; Varanini, Zeno

    2016-01-01

    The induction, i.e., the rapid increase of nitrate ([Formula: see text]) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to [Formula: see text] treatment were characterized in terms of changes in [Formula: see text] uptake rate and plasma membrane (PM) H(+)-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H(+)-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H(+)-ATPase transcripts indicates that those mainly involved in the response to [Formula: see text] treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H(+)-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms.

  12. Time-Resolved Investigation of Molecular Components Involved in the Induction of NO3– High Affinity Transport System in Maize Roots

    PubMed Central

    Pii, Youry; Alessandrini, Massimiliano; Dall’Osto, Luca; Guardini, Katia; Prinsi, Bhakti; Espen, Luca; Zamboni, Anita; Varanini, Zeno

    2016-01-01

    The induction, i.e., the rapid increase of nitrate (NO3–) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to NO3– treatment were characterized in terms of changes in NO3– uptake rate and plasma membrane (PM) H+-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H+-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H+-ATPase transcripts indicates that those mainly involved in the response to NO3– treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H+-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms. PMID:27877183

  13. Downregulating the sucrose transporter VpSUT1 in Verbascum phoeniceum does not inhibit phloem loading

    PubMed Central

    Zhang, Cankui; Turgeon, Robert

    2009-01-01

    Sucrose is loaded into the phloem in the minor veins of leaves before export. Two active, species-specific loading mechanisms have been proposed. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the putative second mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the phloem, not by transporter activity. Several sucrose-transporting plants have been shown to be apoplastic loaders by downregulating sucrose transporter 1 (SUT1), leading to accumulation of sugars and leaf chlorosis. In this study we compared the effect of downregulating SUT1 in Nicotiana tabacum, a sucrose transporter, and Verbascum phoeniceum, a species that transports raffinose and stachyose. To test the effectiveness of RNAi downregulation, we measured SUT1 mRNA levels and sucrose-H+ symport in leaf discs. Mild NtSUT1 downregulation in N. tabacum resulted in the pronounced phenotype associated with loading inhibition. In contrast, no such phenotype developed when VpSUT1 was downregulated in V. phoeniceum, leaving minimal sucrose transport activity. Only those plants with the most severe VpSUT1 downregulation accumulated more carbohydrate than usual and these plants were normal by other criteria: growth rate, photosynthesis, and ability to clear starch during the night. The results provide direct evidence that the mechanism of phloem loading in V. phoeniceum does not require active sucrose uptake from the apoplast and strongly supports the conclusion that the loading pathway is symplastic in this species. PMID:19846784

  14. Transport and sorting of the solanum tuberosum sucrose transporter SUT1 is affected by posttranslational modification.

    PubMed

    Krügel, Undine; Veenhoff, Liesbeth M; Langbein, Jennifer; Wiederhold, Elena; Liesche, Johannes; Friedrich, Thomas; Grimm, Bernhard; Martinoia, Enrico; Poolman, Bert; Kühn, Christina

    2008-09-01

    The plant sucrose transporter SUT1 from Solanum tuberosum revealed a dramatic redox-dependent increase in sucrose transport activity when heterologously expressed in Saccharomyces cerevisiae. Plant plasma membrane vesicles do not show any change in proton flux across the plasma membrane in the presence of redox reagents, indicating a SUT1-specific effect of redox reagents. Redox-dependent sucrose transport activity was confirmed electrophysiologically in Xenopus laevis oocytes with SUT1 from maize (Zea mays). Localization studies of green fluorescent protein fusion constructs showed that an oxidative environment increased the targeting of SUT1 to the plasma membrane where the protein concentrates in 200- to 300-nm raft-like microdomains. Using plant plasma membranes, St SUT1 can be detected in the detergent-resistant membrane fraction. Importantly, in yeast and in plants, oxidative reagents induced a shift in the monomer to dimer equilibrium of the St SUT1 protein and increased the fraction of dimer. Biochemical methods confirmed the capacity of SUT1 to form a dimer in plants and yeast cells in a redox-dependent manner. Blue native PAGE, chemical cross-linking, and immunoprecipitation, as well as the analysis of transgenic plants with reduced expression of St SUT1, confirmed the dimerization of St SUT1 and Sl SUT1 (from Solanum lycopersicum) in planta. The ability to form homodimers in plant cells was analyzed by the split yellow fluorescent protein technique in transiently transformed tobacco (Nicotiana tabacum) leaves and protoplasts. Oligomerization seems to be cell type specific since under native-like conditions, a phloem-specific reduction of the dimeric form of the St SUT1 protein was detectable in SUT1 antisense plants, whereas constitutively inhibited antisense plants showed reduction only of the monomeric form. The role of redox control of sucrose transport in plants is discussed.

  15. High-affinity glutamate transporter and glutamine synthetase content in longissimus dorsi and adipose tissues of growing Angus steers differs among suckling, weanling, backgrounding, and finishing production stages.

    PubMed

    Matthews, J C; Huang, J; Rentfrow, G

    2016-03-01

    Skeletal muscle and adipose tissues play important roles in maintaining whole-body Glu and N homeostasis by the uptake of Glu and release of Gln. To test the hypothesis that expression of high-affinity Glu transporters (GLAST1, EAAT4, EAAC1, GLT-1) and glutamine synthetase (GS) would increase in longissimus dorsi and adipose tissue of newborn Angus steers randomly assigned ( = 6) to develop through suckling (S; 32 d) and/or weanling (W; 184 d), backgrounding (B; 248 d), and finishing (F; 423 d) production stages. Carcass quality was determined at slaughter to verify shifts in adipose and lean deposition with development. Expression of mRNA (RT-PCR/Southern) and relative protein abundance (Western analysis) were determined in tissue homogenates isolated from longissimus dorsi, and kidney and subcutaneous adipose. The effect of production stage or tissue type on carcass and protein abundance was assessed by 1-way ANOVA using the GLM procedure of SAS, and Fisher's protected LSD procedure was used to separate data means. Neither GLAST1 nor EAAT4 mRNA or protein was detected. EAAC1, GLT-1, and GS mRNA were identified in all tissues, but GLT-1 and GS protein were not detected in kidney or subcutaneous adipose, and GS protein was not detected in longissimus dorsi. The EAAC1 content of subcutaneous ( = 0.06) and kidney ( = 0.02) adipose was 2 times greater in B and F than W steers, whereas GS was 5 times greater ( < 0.07) in B than F steers (B = W > F). For longissimus dorsi, EAAC1 ( < 0.01) and GLT-1 ( < 0.04) content decreased with development (S > W > B = F, S = W > B = F, respectively). Within F steers, EAAC1 and GLT-1 mRNA was expressed by subcutaneous, kidney, omental, mesenchymal, and intramuscular adipose tissues, whereas GS mRNA was expressed by all except for intramuscular. Only EAAC1 protein was detected in any adipose tissue, with EAAC1 content being 104% and 112% greater ( < 0.01) in intramuscular than in kidney or subcutaneous adipose, respectively, and not

  16. Are sucrose transporter expression profiles linked with patterns of biomass partitioning in Sorghum phenotypes?

    PubMed Central

    Milne, Ricky J.; Byrt, Caitlin S.; Patrick, John W.; Grof, Christopher P. L.

    2013-01-01

    Sorghum bicolor is a genetically diverse C4 monocotyledonous species, encompassing varieties capable of producing high grain yields as well as sweet types which accumulate soluble sugars (predominantly sucrose) within their stems to high concentrations. Sucrose produced in leaves (sources) enters the phloem and is transported to regions of growth and storage (sinks). It is likely that sucrose transporter (SUT) proteins play pivotal roles in phloem loading and the delivery of sucrose to growth and storage sinks in all Sorghum ecotypes. Six SUTs are present in the published Sorghum genome, based on the BTx623 grain cultivar. Homologues of these SUTs were cloned and sequenced from the sweet cultivar Rio, and compared with the publically available genome information. SbSUT5 possessed nine amino acid sequence differences between the two varieties. Two of the remaining five SUTs exhibited single variations in their amino acid sequences (SbSUT1 and SbSUT2) whilst the rest shared identical sequences. Complementation of a mutant Saccharomyces yeast strain (SEY6210), unable to grow upon sucrose as the sole carbon source, demonstrated that the Sorghum SUTs were capable of transporting sucrose. SbSUT1, SbSUT4, and SbSUT6 were highly expressed in mature leaf tissues and hence may contribute to phloem loading. In contrast, SbSUT2 and SbSUT5 were expressed most strongly in sinks consistent with a possible role of facilitating sucrose import into stem storage pools and developing inflorescences. PMID:23805151

  17. Are sucrose transporter expression profiles linked with patterns of biomass partitioning in Sorghum phenotypes?

    PubMed

    Milne, Ricky J; Byrt, Caitlin S; Patrick, John W; Grof, Christopher P L

    2013-01-01

    Sorghum bicolor is a genetically diverse C4 monocotyledonous species, encompassing varieties capable of producing high grain yields as well as sweet types which accumulate soluble sugars (predominantly sucrose) within their stems to high concentrations. Sucrose produced in leaves (sources) enters the phloem and is transported to regions of growth and storage (sinks). It is likely that sucrose transporter (SUT) proteins play pivotal roles in phloem loading and the delivery of sucrose to growth and storage sinks in all Sorghum ecotypes. Six SUTs are present in the published Sorghum genome, based on the BTx623 grain cultivar. Homologues of these SUTs were cloned and sequenced from the sweet cultivar Rio, and compared with the publically available genome information. SbSUT5 possessed nine amino acid sequence differences between the two varieties. Two of the remaining five SUTs exhibited single variations in their amino acid sequences (SbSUT1 and SbSUT2) whilst the rest shared identical sequences. Complementation of a mutant Saccharomyces yeast strain (SEY6210), unable to grow upon sucrose as the sole carbon source, demonstrated that the Sorghum SUTs were capable of transporting sucrose. SbSUT1, SbSUT4, and SbSUT6 were highly expressed in mature leaf tissues and hence may contribute to phloem loading. In contrast, SbSUT2 and SbSUT5 were expressed most strongly in sinks consistent with a possible role of facilitating sucrose import into stem storage pools and developing inflorescences.

  18. The Trehalose Phosphotransferase System (PTS) in E. coli W Can Transport Low Levels of Sucrose that Are Sufficient to Facilitate Induction of the csc Sucrose Catabolism Operon

    PubMed Central

    Steen, Jennifer A.; Bohlke, Nina; Vickers, Claudia E.; Nielsen, Lars K.

    2014-01-01

    Plasticity in substrate acceptance is a well-characterised phenomenon for disaccharide transporters. Sucrose, a non-reducing disaccharide, is usually metabolised via either the permease-mediated chromosomally-encoded sucrose catabolism (csc) regulon or the sucrose phosphotransferase system (PTS). E. coli W is a fast-growing strain which efficiently utilises sucrose at concentrations above 1% via the csc regulon. To examine if sucrose could be metabolised via other routes, a library of transposon mutants was generated and screened on 0.2% sucrose. One mutant identified from this library had an insertion in the repressor for the regulon controlling catabolism of the disaccharide trehalose (treR). A series of mutants was constructed to elucidate the mechanism of sucrose utilization in the treR insertion strain. Analysis of these mutants provided evidence that deletion of TreR enables uptake of sucrose via TreB, an enzyme II protein required for PTS-mediated uptake of trehalose. Once inside the cell, this sucrose is not processed by the TreC hydrolase, nor is it sufficient for growth of the strain. QRT-PCR analysis showed that levels of cscA (invertase) transcript increased in the WΔtreR mutant relative to the wild-type strain when grown under low sucrose conditions. This result suggests that the intracellular sucrose provided by TreB can facilitate de-repression of the csc regulon, leading to increased gene expression, sucrose uptake and sucrose utilization in the treR mutant. PMID:24586369

  19. Sucrose Transporter ZmSut1 Expression and Localization Uncover New Insights into Sucrose Phloem Loading1[OPEN

    PubMed Central

    Baker, R. Frank; Leach, Kristen A.; Boyer, Nathanial R.; Skopelitis, Tara; Jackson, David; Braun, David M.

    2016-01-01

    Sucrose transporters (SUTs) translocate sucrose (Suc) across cellular membranes, and in eudicots, multiple SUTs are known to function in Suc phloem loading in leaves. In maize (Zea mays), the Sucrose Transporter1 (ZmSut1) gene has been implicated in Suc phloem loading based upon RNA expression in leaves, electrophysiological experiments, and phenotypic analysis of zmsut1 mutant plants. However, no previous studies have examined the cellular expression of ZmSut1 RNA or the subcellular localization of the ZmSUT1 protein to assess the gene’s hypothesized function in Suc phloem loading or to evaluate its potential roles, such as phloem unloading, in nonphotosynthetic tissues. To this end, we performed RNA in situ hybridization experiments, promoter-reporter gene analyses, and ZmSUT1 localization studies to elucidate the cellular expression pattern of the ZmSut1 transcript and protein. These data showed that ZmSut1 was expressed in multiple cell types throughout the plant and indicated that it functions in phloem companion cells to load Suc and also in other cell types to retrieve Suc from the apoplasm to prevent its accumulation and loss to the transpiration stream. Additionally, by comparing a phloem-mobile tracer with ZmSut1 expression, we determined that developing maize leaves dynamically switch from symplasmic to apoplasmic phloem unloading, reconciling previously conflicting reports, and suggest that ZmSut1 does not have an apparent function in either unloading process. A model for the dual roles for ZmSut1 function (phloem loading and apoplasmic recycling), Sut1 evolution, and its possible use to enhance Suc export from leaves in engineering C3 grasses for C4 photosynthesis is discussed. PMID:27621426

  20. Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae.

    PubMed

    Bartölke, Rabea; Heinisch, Jürgen J; Wieczorek, Helmut; Vitavska, Olga

    2014-12-01

    The members of the solute carrier 45 (SLC45) family have been implicated in the regulation of glucose homoeostasis in the brain (SLC45A1), with skin and hair pigmentation (SLC45A2), and with prostate cancer and myelination (SLC45A3). However, apart from SLC45A1, a proton-associated glucose transporter, the function of these proteins is still largely unknown, although sequence similarities to plant sucrose transporters mark them as a putative sucrose transporter family. Heterologous expression of the three members SLC45A2, SLC45A3 and SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose transporters. [(14)C]Sucrose-uptake measurements revealed intermediate transport affinities with Km values of approximately 5 mM. Transport activities were best under slightly acidic conditions and were inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, demonstrating an H(+)-coupled transport mechanism. Na(+), on the other hand, had no effect on sucrose transport. Competitive inhibition assays indicated a possible transport also of glucose and fructose. Real-time PCR of mouse tissues confirmed mRNA expression of SLC45A2 in eyes and skin and of SLC45A3 primarily in the prostate, but also in other tissues, whereas SLC45A4 showed a predominantly ubiquitous expression. Altogether the results provide new insights into the physiological significance of SLC45 family members and challenge existing concepts of mammalian sugar transport, as they (i) transport a disaccharide, and (ii) perform secondary active transport in a proton-dependent manner.

  1. Sucrose transport and phloem unloading in peach fruit: potential role of two transporters localized in different cell types.

    PubMed

    Zanon, Laura; Falchi, Rachele; Santi, Simonetta; Vizzotto, Giannina

    2015-06-01

    Several complex physiological processes, which include long-distance translocation in the phloem and unloading in sink tissues, govern the partitioning of sugars in economically important organs, such as peach fruit. In this study, we took advantage of a symplastic tracer, carboxyfluorescein (CF), providing evidence for an apoplastic sucrose transfer in the early (SI) and middle (SIII) phases of peach fruit development. Moreover, using a combination of in situ hybridization and laser microdissection-assisted expression analysis, three putative sucrose transporters encoding genes (PpSUT1, PpSUT2, PpSUT4) were transcriptionally analyzed to relate their expression with sucrose storage in this organ. Our study revealed that PpSUT2 and PpSUT4 are the genes predominantly expressed in fruit flesh, and the detailed analysis of their expression pattern in the different cell types enabled us to suggest a specialized role in sucrose distribution. Both PpSUTs transporters could be involved in the retrieval of sucrose lost from the symplastic continuum of the phloem and, when expressed in parenchyma cells, they could be active in the import of sucrose into sink tissues, via symport from the apoplast. An alternative hypothesis has been proposed and discussed for PpSUT4 because of its putative tonoplastic localization. Taken together, our results provide new insights into the molecular mechanisms underpinning sucrose unloading and accumulation in peach fruit.

  2. The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response1[OPEN

    PubMed Central

    Brauer, Elizabeth K.; Ahsan, Nagib; Kato, Naohiro; Coluccio, Alison E.; Thelen, Jay J.

    2016-01-01

    Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca2+ uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H+)/K+ symporter that mediates a high-affinity uptake during K+ deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K+ efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K+ loss. Taken together, our results position ILK1 as a link between plant defense pathways and K+ homeostasis. PMID:27208244

  3. In vivo transport of three radioactive [(18)F]-fluorinated deoxysucrose analogs by the maize sucrose transporter ZmSUT1.

    PubMed

    Tran, Thu M; Hampton, Carissa S; Brossard, Tom W; Harmata, Michael; Robertson, J David; Jurisson, Silvia S; Braun, David M

    2017-03-08

    Sucrose transporter (SUT) proteins translocate sucrose across cell membranes; however, mechanistic aspects of sucrose binding by SUTs are not well resolved. Specific hydroxyl groups in sucrose participate in hydrogen bonding with SUT proteins. We previously reported that substituting a radioactive fluorine-18 [(18)F] at the C-6' position within the fructosyl moiety of sucrose did not affect sucrose transport by the maize (Zea mays) ZmSUT1 protein. To determine how (18)F substitution of hydroxyl groups at two other positions within sucrose, the C-1' in the fructosyl moiety or the C-6 in the glucosyl moiety, impact sucrose transport, we synthesized 1'-[F(18)]fluoro-1'-deoxysucrose and 6-[F(18)]fluoro-6-deoxysucrose ([(18)F]FDS) analogs. Each [(18)F]FDS derivative was independently introduced into wild-type or sut1 mutant plants, which are defective in sucrose phloem loading. All three (1'-, 6'-, and 6-) [(18)F]FDS derivatives were efficiently and equally translocated, similarly to carbon-14 [(14)C]-labeled sucrose. Hence, individually replacing the hydroxyl groups at these positions within sucrose does not interfere with substrate recognition, binding, or membrane transport processes, and hydroxyl groups at these three positions are not essential for hydrogen bonding between sucrose and ZmSUT1. [(18)F]FDS imaging afforded several advantages compared to [(14)C]-sucrose detection. We calculated that 1'-[(18)F]FDS was transported at approximately a rate of 0.90 ± 0.15 m.h-1 in wild-type leaves, and at 0.68 ± 0.25 m.h-1 in sut1 mutant leaves. Collectively, our data indicated that [(18)F]FDS analogs are valuable tools to probe sucrose-SUT interactions and to monitor sucrose transport in plants.

  4. Sucrose transport and phloem unloading in stem of Vicia faba: possible involvement of a sucrose carrier and osmotic regulation

    SciTech Connect

    Aloni, B.; Wyse, R.E.; Griffith, S.

    1986-06-01

    After pulse labeling of a source leaf with /sup 14/CO/sub 2/, stem sections of Vicia faba plants were cut and the efflux characteristics of /sup 14/C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of /sup 14/C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of (/sup 14/C)sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced (/sup 14/C)sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved.

  5. SUT Sucrose and MST Monosaccharide Transporter Inventory of the Selaginella Genome

    PubMed Central

    Lalonde, Sylvie; Frommer, Wolf B.

    2012-01-01

    Most metazoa use hexose transporters to acquire hexoses from their diet and as a transport form for distributing carbon and energy within their bodies; insects use trehalose, and plants use sucrose as their major form for translocation. Plant genomes contain at least three families of mono- and disaccharide transporters: monosaccharide/polyol transporters that are evolutionary closely related to the yeast and human glucose transporters, sucrose transporters of the SUT family, which similar to the hexose transporters belong to the major facilitator superfamily, but share only minimal amino acid sequence homology with the hexose transporters, and the family of SWEET sugar transporters conserved between animals and plants. Recently, the genome sequence of the spikemoss Selaginella has been determined. In order to study the evolution of sugar transport in plants, we carefully annotated of the complement of sugar transporters in Selaginella. We review the current knowledge regarding sugar transport in spikemoss and provide phylogenetic analyses of the complement of MST and SUT homologs in Selaginella (and Physcomitrella). PMID:22645575

  6. Activation of sucrose transport in defoliated Lolium perenne L.: an example of apoplastic phloem loading plasticity.

    PubMed

    Berthier, Alexandre; Desclos, Marie; Amiard, Véronique; Morvan-Bertrand, Annette; Demmig-Adams, Barbara; Adams, William W; Turgeon, Robert; Prud'homme, Marie-Pascale; Noiraud-Romy, Nathalie

    2009-07-01

    The pathway of carbon phloem loading was examined in leaf tissues of the forage grass Lolium perenne. The effect of defoliation (leaf blade removal) on sucrose transport capacity was assessed in leaf sheaths as the major carbon source for regrowth. The pathway of carbon transport was assessed via a combination of electron microscopy, plasmolysis experiments and plasma membrane vesicles (PMVs) purified by aqueous two-phase partitioning from the microsomal fraction. Results support an apoplastic phloem loading mechanism. Imposition of an artificial proton-motive force to PMVs from leaf sheaths energized an active, transient and saturable uptake of sucrose (Suc). The affinity of Suc carriers for Suc was 580 microM in leaf sheaths of undefoliated plants. Defoliation induced a decrease of K(m) followed by an increase of V(max). A transporter was isolated from stubble (including leaf sheaths) cDNA libraries and functionally expressed in yeast. The level of L.perenne SUcrose Transporter 1 (LpSUT1) expression increased in leaf sheaths in response to defoliation. Taken together, the results indicate that Suc transport capacity increased in leaf sheaths of L. perenne in response to leaf blade removal. This increase might imply de novo synthesis of Suc transporters, including LpSUT1, and may represent one of the mechanisms contributing to rapid refoliation.

  7. Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumour suppressor in colon that protects against colitis and colon cancer under low-fibre dietary conditions.

    PubMed

    Gurav, Ashish; Sivaprakasam, Sathish; Bhutia, Yangzom D; Boettger, Thomas; Singh, Nagendra; Ganapathy, Vadivel

    2015-07-15

    Mammalian colon harbours trillions of bacteria under physiological conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. In the present study we show that Slc5a8 (solute carrier gene family 5a, member 8), a Na(+)-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T-cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naive T-cells into immunosuppressive forkhead box P3(+) (FoxP3(+)) Tregs (regulatory T-cells) and suppress conversion of naive T-cells into pro-inflammatory interferon (IFN)-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T-cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T-cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fibre intake but not when dietary fibre intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fibre and mucosal immune system via the bacterial metabolite butyrate and that this transporter is a conditional tumour suppressor in colon linked to dietary fibre content.

  8. Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumor suppressor in colon that protects against colitis and colon cancer under low-fiber dietary conditions

    PubMed Central

    Gurav, Ashish; Sivaprakasam, Sathish; Bhutia, Yangzom D.; Boettger, Thomas; Singh, Nagendra; Ganapathy, Vadivel

    2016-01-01

    Summary Mammalian colon harbors trillions of bacteria under physiologic conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. Here we show that Slc5a8, a Na+-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naïve T cells into immunosuppressive FoxP3+ Tregs, and suppress conversion of naïve T cells into pro-inflammatory IFN-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fiber intake but not when dietary fiber intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fiber and mucosal immune system via the bacterial metabolite butyrate, and that this transporter is a conditional tumor suppressor in colon linked to dietary fiber content. PMID:25984582

  9. Partitioning and transport of the translocates mannitol and sucrose in the light and dark in celery (Apium Graveolens L. )

    SciTech Connect

    Davis, J.M.; Loescher, W.H.

    1987-04-01

    Sucrose and mannitol are major photosynthetic products and translocates in celery. Assimilate partitioning and transport were studied by pulse-labeling leaves with /sup 14/CO/sub 2/ followed by different length chases in ambient air. After a 2 h chase in the light there was more /sup 14/C in sucrose than mannitol in source leaves and their petioles. In contrast after a 2 h dark chase leaves contained more /sup 14/C in mannitol than sucrose but petioles had more /sup 14/C in sucrose than mannitol. After a 15 h chase (6 h light; 9 h dark) labeled sucrose was higher in source petiole vascular bundles than in adjacent parenchyma tissue but label in glucose and fructose was higher in the parenchyma tissue. After the 15 h chase most of the /sup 14/C remaining in developing sink leaves and their petioles was in mannitol. Although in the light mannitol:sucrose ratios are the same in leaf and petiole tissues, in the dark sucrose is initially the major translocate with mannitol becoming more important as leaf sucrose pools are depleted. When synthesized, sucrose is rapidly transported and then metabolized to hexose sugars whereas mannitol is used both for transport and storage.

  10. Characterisation of [11C]PR04.MZ in Papio anubis baboon: A selective high-affinity radioligand for quantitative imaging of the dopamine transporter

    SciTech Connect

    Riss P. J.; Fowler J.; Riss, P.J.; Hooker, J.M.; Shea, C.; Xu, Y.; Carter, P.; Warner, D.; Ferrari V.; Kim, S.W.; Aigbirhio, F.I.; Fowler, J.S.; Roesch, F.

    2011-10-25

    N-(4-fluorobut-2-yn-1-yl)-2{beta}-carbomethoxy-3{beta}-(4{prime}-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [{sup 11}C]PR04.MZ ([{sup 11}C]-1) has been developed using GMP compliant equipment. An adult female Papioanubis baboon was studied using a test-retest protocol with [{sup 11}C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (fP), plasma input functions and metabolic degradation of the radiotracer [{sup 11}C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (VT) and non-displaceable binding potentials (BPND) for various brain regions and the blood were obtained from kinetic modelling. [{sup 11}C]-1 shows promising results as aselective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man.

  11. Transcriptional coordination and abscisic acid mediated regulation of sucrose transport and sucrose-to-starch metabolism related genes during grain filling in wheat (Triticum aestivum L.).

    PubMed

    Mukherjee, Shalini; Liu, Aihua; Deol, Kirandeep K; Kulichikhin, Konstanin; Stasolla, Claudio; Brûlé-Babel, Anita; Ayele, Belay T

    2015-11-01

    Combining physiological, molecular and biochemical approaches, this study investigated the transcriptional coordination and abscisic acid (ABA) mediated regulation of genes involved in sucrose import and its conversion to starch during grain filling in wheat. Sucrose import appears to be mediated by seed localized TaSUT1, mainly TaSUT1D, while sucrose cleavage by TaSuSy2. Temporal overlapping of the transcriptional activation of AGPL1 and AGPS1a that encode AGPase with that of the above genes suggests their significance in the synthesis of ADP-glucose; TaAGPL1A and TaAGPL1D contributing the majority of AGPL1 transcripts. ABA induced repressions of TaSUT1, TaSuSy2, TaAGPL1 and TaAGPS1a imply that ABA negatively regulates sucrose import into the endosperm and its subsequent metabolism to ADP-glucose, the substrate for starch synthesis. The formations of amyloses and amylopectin from ADP-glucose appear to be mediated by specific members of GBSS, and SS, SBE and DBE gene families, and the ABA-induced transcriptional change in most of these genes implies that ABA regulates amylose and amylopectin synthesis. The findings provide insights into the molecular mechanisms underlying the coordination and ABA mediated regulation of sucrose transport into the developing endosperm and its subsequent metabolism to starch during grain filling in wheat.

  12. MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity.

    PubMed

    Lu, Shuo; Zgurskaya, Helen I

    2013-11-01

    The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC.

  13. Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification[W

    PubMed Central

    Krügel, Undine; Veenhoff, Liesbeth M.; Langbein, Jennifer; Wiederhold, Elena; Liesche, Johannes; Friedrich, Thomas; Grimm, Bernhard; Martinoia, Enrico; Poolman, Bert; Kühn, Christina

    2008-01-01

    The plant sucrose transporter SUT1 from Solanum tuberosum revealed a dramatic redox-dependent increase in sucrose transport activity when heterologously expressed in Saccharomyces cerevisiae. Plant plasma membrane vesicles do not show any change in proton flux across the plasma membrane in the presence of redox reagents, indicating a SUT1-specific effect of redox reagents. Redox-dependent sucrose transport activity was confirmed electrophysiologically in Xenopus laevis oocytes with SUT1 from maize (Zea mays). Localization studies of green fluorescent protein fusion constructs showed that an oxidative environment increased the targeting of SUT1 to the plasma membrane where the protein concentrates in 200- to 300-nm raft-like microdomains. Using plant plasma membranes, St SUT1 can be detected in the detergent-resistant membrane fraction. Importantly, in yeast and in plants, oxidative reagents induced a shift in the monomer to dimer equilibrium of the St SUT1 protein and increased the fraction of dimer. Biochemical methods confirmed the capacity of SUT1 to form a dimer in plants and yeast cells in a redox-dependent manner. Blue native PAGE, chemical cross-linking, and immunoprecipitation, as well as the analysis of transgenic plants with reduced expression of St SUT1, confirmed the dimerization of St SUT1 and Sl SUT1 (from Solanum lycopersicum) in planta. The ability to form homodimers in plant cells was analyzed by the split yellow fluorescent protein technique in transiently transformed tobacco (Nicotiana tabacum) leaves and protoplasts. Oligomerization seems to be cell type specific since under native-like conditions, a phloem-specific reduction of the dimeric form of the St SUT1 protein was detectable in SUT1 antisense plants, whereas constitutively inhibited antisense plants showed reduction only of the monomeric form. The role of redox control of sucrose transport in plants is discussed. PMID:18790827

  14. Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior

    PubMed Central

    Öner-Sieben, Soner; Rappl, Christine; Sauer, Norbert; Stadler, Ruth; Lohaus, Gertrud

    2015-01-01

    Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker’s yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior. PMID:26022258

  15. Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior.

    PubMed

    Öner-Sieben, Soner; Rappl, Christine; Sauer, Norbert; Stadler, Ruth; Lohaus, Gertrud

    2015-08-01

    Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker's yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior.

  16. High-affinity K+ uptake in pepper plants.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2005-06-01

    High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

  17. Transport and metabolism of a sucrose analog (1'-fluorosucrose) into Zea mays L. Endosperm without invertase hydrolysis

    SciTech Connect

    Schmalstig, J.G.; Hitz, W.D.

    1987-12-01

    1'-fluorosucrose (FS), a sucrose analog resistant to hydrolysis by invertase, was transported from husk leaves into maize (Zea mays L.) kernels with the same magnitude and kinetics as sucrose. /sup 14/C-Label from (/sup 14/C)FS and (/sup 14/C)sucrose in separate experiments was distributed similarly between the pedicel, endosperm, and embryo with time. FS passed through maternal tissue and was adsorbed intact into the endosperm where it was metabolized and used in synthesis of sucrose and methanol-chloroform-water insolubles. Accumulation of (/sup 14/C)sucrose from supplied (/sup 14/C)glucosyl-FS indicated that the glucose moiety from the breakdown of sucrose (here FS), which normally occurs in the process of starch synthesis in maize endosperm, was available to the pool of substrates for resynthesis of sucrose. Uptake of FS into maize endosperm without hydrolysis suggest that despite the presence of invertase in maternal tissues and the hydrolysis of a large percentage of sucrose unloaded form the phloem, hexoses are not specifically needed for uptake into maize endosperm.

  18. Cloning and expression analysis of PpSUT2 encoding a sucrose transporter in pear.

    PubMed

    Tang, J; Lin, J; Zhang, B L; Wang, Z H; Li, X G; Chang, Y H

    2014-10-31

    A 1794-bp cDNA fragment was amplified from mRNA isolated from pear (Pyrus pyrifolia NaKai. Cuiguan) leaves by using primers based on the sequences generated during the analysis of the pear transcriptome. The 597-amino acid sequence encoded by the cDNA was compared with the sequences in GenBank, and it was found to be similar to that of members of the sucrose-proton co-transporter family. The hydrophobic protein, which was predicted to have 11 transmembrane domains, was designated as PpSUT2. Real-time fluorescent quantitative polymerase chain reaction analysis indicated the accumulation of PpSUT2 mRNA throughout the plant, with the highest levels in the buds. Analysis of the expression of PpSUT2 during fruit development showed that the abundance of its transcripts increased at the end of April and then decreased to the lowest level at the end of July. Subcellular localization studies with the pCXDG vector as a probe demonstrated that PpSUT2 localized to cell membranes. An expression vector was constructed by inserting the PpSUT2 cDNA into pET32(a), and the vector was expressed in Escherichia coli (strain BL21) after induction with 1 mM isopropyl b-d-1-thiogalactopyranoside at 25°C. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified the induction of a 71-kDa protein. Further analysis indicated that PpSUT2 might be not directly involved in sucrose transport, instead, functioning as a sucrose sensor on the cytoplasmic membrane.

  19. Essential role of K(+) uptake permease (Kup) for resistance to sucrose-induced stress in Gluconacetobacter diazotrophicus PAl 5.

    PubMed

    de Oliveira, Marcos V V; Intorne, Aline C; Vespoli, Luciano de S; Andrade, Leandro F; Pereira, Leandro de M; Rangel, Patrícia L; de Souza Filho, Gonçalo A

    2017-04-01

    Microorganisms are constantly challenged by stressful conditions, such as sugar-rich environments. Such environments can cause an imbalance of biochemical activities and compromise cell multiplication. Gluconacetobacter diazotrophicus PAl 5 is among the most sugar-tolerant bacteria, capable of growing in the presence of up to 876 mM sucrose. However, the molecular mechanisms involved in its response to high sucrose remain unknown. The present work aimed to identify sucrose-induced stress resistance genes in G. diazotrophicus PAl 5. Screening of a Tn5 transposon insertion library identified a mutant that was severely compromised in its resistance to high sucrose concentrations. Molecular characterization revealed that the mutation affected the kupA gene, which encodes a K(+) uptake transporter (KupA). Functional complementation of the mutant with the wild type kupA gene recovered the sucrose-induced stress resistance phenotype. High sucrose resistance assay, under different potassium concentrations, revealed that KupA acts as a high-affinity K(+) transporter, which is essential for resistance to sucrose-induced stress, when extracellular potassium levels are low. This study is the first to show the essential role of the KupA protein for resistance to sucrose-induced stress in bacteria by acting as a high-affinity potassium transporter in G. diazotrophicus PAl 5.

  20. Identification and functional assay of the interaction motifs in the partner protein OsNAR2.1 of the two-component system for high-affinity nitrate transport.

    PubMed

    Liu, Xiaoqin; Huang, Daimin; Tao, Jinyuan; Miller, Anthony J; Fan, Xiaorong; Xu, Guohua

    2014-10-01

    A partner protein, NAR2, is essential for high-affinity nitrate transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and nitrate transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 ((15)N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.

  1. Molecular cloning and expression analysis of a gene for sucrose transporter from pear (Pyrus bretschneideri Rehd.) fruit.

    PubMed

    Zhang, Huping; Zhang, Shujun; Qin, Gaihua; Wang, Lifen; Wu, Tao; Qi, Kaijie; Zhang, Shaoling

    2013-12-01

    Here we report the cloning of a sucrose transporter cDNA from pear (Pyrus bretschneideri Rehd. cv 'Yali') fruit and an analysis of the expression of the gene. A cDNA clone, designated PbSUT1 was identified as a sucrose transporter cDNA from its sequence homology at the amino acid level to sucrose transporters that have been cloned from other higher plant species. PbSUT1 potentially encoded a protein of 499 amino acid residues with a predicted molecular mass of 53.4 kDa and an isoelectric point (pI) of 9.21. Phylogenetic analysis revealed that the PbSUT1 belonged to type III SUTs and was more closely related to the MdSUT1 from apple fruit. Some major facilitator superfamily (MFS)-specific sequence motifs were found in the predicted PbSUT1 peptides, and an MFS_1 domain was located at the amino acid positions of 29-447 of the sequence. A study of gene expression along fruit development showed that PbSUT1 transcripts are present at all stages but significantly increase before fruit enlargement and during the ripening process with increasing sucrose levels. In contrast, the expression levels don't change much during the period of rapid fruit growth. This work shows that sucrose transporter may play a role in the accumulation of sugars during maturation and in maintaining the internal cellular distribution.

  2. Synthesis and molecular modeling of new 1-aryl-3-[4-arylpiperazin-1-yl]-1-propane derivatives with high affinity at the serotonin transporter and at 5-HT(1A) receptors.

    PubMed

    Orús, Lara; Pérez-Silanes, Silvia; Oficialdegui, Ana-M; Martínez-Esparza, Javier; Del Castillo, Juan-C; Mourelle, Marisa; Langer, Thierry; Guccione, Salvatore; Donzella, Giuseppina; Krovat, Eva M; Poptodorov, Konstantin; Lasheras, Berta; Ballaz, Santiago; Hervías, Isabel; Tordera, Rosa; Del Río, Joaquín; Monge, Antonio

    2002-09-12

    It has been proposed that 5-HT(1A) receptor antagonists augment the antidepressant efficacy of selective serotonin (5-HT) reuptake inhibitors. In a search toward new and efficient antidepressants, 1-(aryl)-3-[4-arylpiperazin-1-yl]-1-propane molecular hybrids were designed, synthesized, and evaluated for 5-HT reuptake inhibition and 5-HT(1A) receptor affinity. The design was based in coupling structural moieties related to inhibition of serotonin reuptake, such as benzo[b]thiophene derivatives to arylpiperazines, typical 5-HT(1A) receptor ligands. In binding studies, several compounds showed affinity at the 5-HT transporter and at 5-HT(1A) receptors. Molecular modeling studies predicted the pharmacophore elements required for high affinity binding and the features that enable to discriminate between agonist, partial agonist, or antagonist action at 5-HT(1A) receptors and 5-HT transporter inhibition. Solvent interactions in desolvation prior to the binding step along with enthalpy and enthropy compensations might be responsible to explain agonist, partial agonist, and antagonist character. Hydrogen-bonding capability seems to be important to break hydrogen interhelical hydrogen bonds or alternatively to form other bonds upon ligand binding. Partial agonists and antagonists are unable to do this as the full agonist, which interacts closely by long-range forces or directly. The compounds showing the higher affinity at both the 5-HT transporter (K(i) < 50 nM) and the 5-HT(1A) receptors (K(i) < 20 nM) were further explored for their ability to stimulate [(35)S]GTPgammaS binding or to antagonize 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT)-stimulated [(35)]GTPgammaS binding to rat hippocampal membranes, an index of agonist/antagonist action at 5-HT(1A) receptors, respectively. Compound 8g exhibited agonist activity (EC(50) = 30 nM) in this assay, whereas compounds 7g and 8h,i behaved as weak partial agonists and 7h-j and 8j,l antagonized the R(+)-8-OH

  3. Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L.

    PubMed

    Li, Fupeng; Wu, Baoduo; Qin, Xiaowei; Yan, Lin; Hao, Chaoyun; Tan, Lehe; Lai, Jianxiong

    2014-08-10

    In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype 'TAS-R8'. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production.

  4. Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

    PubMed Central

    Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui; Harding, Scott A.

    2016-01-01

    Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism in root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability. PMID:27641356

  5. Molecular regulation of sucrose catabolism and sugar transport for development, defence and phloem function.

    PubMed

    Li, Jun; Wu, Limin; Foster, Ryan; Ruan, Yong-Ling

    2017-03-17

    Sucrose (Suc) is the major end product of photosynthesis in mesophyll cells of most vascular plants. It is loaded into phloem of mature leaves for long-distance translocation to non-photosynthetic organs where it is unloaded for diverse uses. Clearly, Suc transport and metabolism is central to plant growth and development and the functionality of the entire vascular system. Despite of vast information in the literature about the physiological roles of individual sugar metabolic enzymes and transporters, there is a lack of systematic evaluation about their molecular regulation from transcriptional to post-translational levels. Knowledge on this topic is essential for understanding and improving plant development, optimising resource distribution and increasing crop productivity. We therefore focused our analyses on molecular control of key players in Suc metabolism and transport, including (i) the identification of promoter elements responsive to sugars and hormones or targeted by transcription factors and microRNAs degrading transcripts of target genes and (ii) modulation of enzyme and transporter activities through protein-protein interactions and other post-translational modifications. We highlighted major remaining questions and discussed opportunities to exploit current understanding to gain new insights into molecular control of carbon partitioning for improving plant performance.

  6. Sucrose Transporter Localization and Function in Phloem Unloading in Developing Stems1[OPEN

    PubMed Central

    Rae, Anne L.; Reinders, Anke

    2017-01-01

    How sucrose transporters (SUTs) regulate phloem unloading in monocot stems is poorly understood and particularly so for species storing high Suc concentrations. To this end, Sorghum bicolor SUTs SbSUT1 and SbSUT5 were characterized by determining their transport properties heterologously expressed in yeast or Xenopus laevis oocytes, and their in planta cellular and subcellular localization. The plasma membrane-localized SbSUT1 and SbSUT5 exhibited a strong selectivity for Suc and high Suc affinities in X. laevis oocytes at pH 5—SbSUT1, 6.3 ± 0.7 mm, and SbSUT5, 2.4 ± 0.5 mm Suc. The Suc affinity of SbSUT1 was dependent on membrane potential and pH. In contrast, SbSUT5 Suc affinity was independent of membrane potential and pH but supported high transport rates at neutral pH. Suc transport by the tonoplast localized SbSUT4 could not be detected using yeast or X. laevis oocytes. Across internode development, SUTs, other than SbSUT4, were immunolocalized to sieve elements, while for elongating and recently elongated internodes, SUTs also were detected in storage parenchyma cells. We conclude that apoplasmic Suc unloading from de-energized protophloem sieve elements in meristematic zones may be mediated by reversal of SbSUT1 and/or by uniporting SWEETs. Storage parenchyma localized SbSUT1 and SbSUT5 may accumulate Suc from the stem apoplasms of elongating and recently elongated internodes, whereas SbSUT4 may function to release Suc from vacuoles. Transiting from an apoplasmic to symplasmic unloading pathway as the stem matures, SbSUT1 and SbSUT5 increasingly function in Suc retrieval into metaphloem sieve elements to maintain a high turgor to drive symplasmic unloading by bulk flow. PMID:27986867

  7. Sucrose Concentration Gradients along the Post-Phloem Transport Pathway in the Maternal Tissues of Developing Wheat Grains.

    PubMed Central

    Fisher, D. B.; Wang, N.

    1995-01-01

    Sucrose concentrations were measured in serial frozen sections of the post-phloem transport pathway in developing wheat (Triticum aestivum L.) grains. In normally importing grains, there was an approximately linear concentration gradient along the pathway, with a difference between the ends of the pathway of about 180 mM. This indicates an unusually low resistance for cell-to-cell transport, due perhaps to the large size-exclusion limit for the pathway. However, the existence of concentration gradients raises presently unresolvable questions about the relative contributions of diffusion versus bulk flow to transport within the symplast. The concentration gradient disappeared when sucrose movement ceased (i.e. in excised grains or when endosperm cavities of attached grains were perfused with p-chloromercuribenzene sulfonate [PCMBS] or with 1660 mOsm sorbitol). PCMBS appeared to block solute release into the endosperm cavity, whereas the sorbitol treatment, previously shown to cause localized plasmolysis in the chalaza, appeared to block movement across the chalaza. Sieve element/companion cell unloading appears to be an important control point for assimilate import. The sucrose concentration gradient and, probably, turgor and osmotic gradients are extremely steep there. PCMBS blocked import without affecting the sucrose concentration in the vascular parenchyma around the phloem. Thus, blockage of unloading was more complex than a simple "backing up" of solutes in the vascular parenchyma. PMID:12228615

  8. Functional characterization of a plant-like sucrose transporter from the beneficial fungus Trichoderma virens. Regulation of the symbiotic association with plants by sucrose metabolism inside the fungal cells.

    PubMed

    Vargas, Walter A; Crutcher, Frankie K; Kenerley, Charles M

    2011-02-01

    Sucrose exuded by plants into the rhizosphere is a crucial component for the symbiotic association between the beneficial fungus Trichoderma and plant roots. In this article we sought to identify and characterize the molecular basis of sucrose uptake into the fungal cells. • Several bioinformatics tools enabled us to identify a plant-like sucrose transporter in the genome of Trichoderma virens Gv29-8 (TvSut). Gene expression profiles in the fungal cells were analyzed by Northern blotting and quantitative real-time PCR (qRT-PCR). Biochemical and physiological studies were conducted on Gv29-8 and fungal strains impaired in the expression of TvSut. • TvSut exhibits biochemical properties similar to those described for sucrose symporters from plants. The null expression of tvsut caused a detrimental effect on fungal growth when sucrose was the sole source of carbon in the medium, and also affected the expression of genes involved in the symbiotic association. • Similar to plants, T. virens contains a highly specific sucrose/H(+) symporter that is induced in the early stages of root colonization. Our results suggest an active sucrose transference from the plant to the fungal cells during the beneficial associations. In addition, our expression experiments suggest the existence of a sucrose-dependent network in the fungal cells that regulates the symbiotic association.

  9. AmSUT1, a sucrose transporter in collection and transport phloem of the putative symplastic phloem loader Alonsoa meridionalis.

    PubMed

    Knop, Christian; Stadler, Ruth; Sauer, Norbert; Lohaus, Gertrud

    2004-01-01

    A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80-91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.

  10. Identification of a new sucrose transporter in rye-grass (LpSUT2): effect of defoliation and putative fructose sensing.

    PubMed

    Berthier, Alexandre; Meuriot, Frédéric; Dédaldéchamp, Fabienne; Lemoine, Rémi; Prud'homme, Marie-Pascale; Noiraud-Romy, Nathalie

    2014-11-01

    Rye-grass fast regrowth after defoliation results from an efficient mobilization of C reserves which are transported as sucrose towards regrowing leaves, and which can be supported by one or several sucrose transporters (SUTs) like LpSUT1. Therefore, our objectives were to isolate, identify, characterize and immunolocalize such sucrose transporters. A protein (LpSUT2) showing a twelve spanning trans-membrane domain, extended N terminal and internal cytoplasmic loop, and kinetic properties consistent with well-known sucrose transporters, was isolated and successfully characterized. Along with LpSUT1, it was mainly localized in mesophyll cells of leaf sheaths and elongating leaf bases. These transporters were also found in parenchyma bundle sheath (PBS) cells but they were not detected in the sieve element/companion cell complex of the phloem. Unlike LpSUT1 transcript levels which increased as a response to defoliation in source and sink tissues, LpSUT2 transcript levels were unaffected by defoliation and weakly expressed. Interestingly, sucrose transport by LpSUT2 was inhibited by fructose. LpSUT1 and LpSUT2 appeared to have different functions. LpSUT1 is proposed to play a key role in C storage and mobilization by allowing sucrose transport between PBS and mesophyll cells, depending on the plant C status. LpSUT2 could be involved in sucrose/fructose sensing at sub-cellular level.

  11. Organ and Tissue-specific Sucrose Transporters. Important Hubs in Gene and Metabolite Networks Regulating Carbon Use in Wood-forming Tissues of Populus

    SciTech Connect

    Harding, Scott A.; Tsai, Chung-Jui

    2016-01-04

    The overall project objective was to probe the relationship between sucrose transporters and plant productivity in the biomass for biofuels woody perennial, Populus. At the time the proposal was written, sucrose transporters had already been investigated in many plant model systems, primarily with respect to the export of photosynthate sucrose from source leaves, and the uptake of sucrose in storage organs and seeds. Preliminary findings by the PI found that in Populus, sucrose transporter genes (SUTs) were well expressed in wood-forming tissues that comprise the feedstock for biofuels production. Because sucrose comprises by far the predominant form in which photosynthate is delivered from source organs to sink organs like roots and wood-forming tissues, SUTs control a gate that nominally at least could impact the allocation or partitioning of sucrose for potentially competing end uses like growth (stem biomass) and storage. In addition, water use might be conditioned by the way in which sucrose is distributed throughout the plant, and/or by the way in which sucrose is partitioned intracellularly. Several dozen transgenic lines were produced in year 1 of the project to perturb the expression ratio of multiple plasma membrane (PM) SUTs (intercellular trafficking), versus the single tonoplast membrane (TM) sucrose transporter that effectively regulates intracellular trafficking of sucrose. It was possible to obtain transgenic lines with dual SUT gene knockdown using the 35S promoter, but not the wood-specific TUA1 promoter. By the end of project year 2, a decision was made to work with the 35S plants while archiving the TUA1 plants. The PhD candidate charged with producing the transgenic lines abandoned the project during its second year, substantially contributing to the decision to operate with just the 35S lines. That student’s interests ranged more toward evolutionary topics, and a report on SUT gene evolution was published (Peng et al 2014).

  12. Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

    PubMed

    Nieberl, P; Ehrl, C; Pommerrenig, B; Graus, D; Marten, I; Jung, B; Ludewig, F; Koch, W; Harms, K; Flügge, U-I; Neuhaus, H E; Hedrich, R; Sauer, N

    2017-05-01

    Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the β-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks.

  13. Switching the mode of sucrose utilization by Saccharomyces cerevisiae

    PubMed Central

    Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

    2008-01-01

    Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose

  14. Cadmium inhibits the induction of high-affinity nitrate uptake in maize (Zea mays L.) roots.

    PubMed

    Rizzardo, Cecilia; Tomasi, Nicola; Monte, Rossella; Varanini, Zeno; Nocito, Fabio F; Cesco, Stefano; Pinton, Roberto

    2012-12-01

    Cadmium (Cd) detoxification involves glutathione and phytochelatins biosynthesis: the higher need of nitrogen should require increased nitrate (NO(3)(-)) uptake and metabolism. We investigated inducible high-affinity NO(3)(-) uptake across the plasma membrane (PM) in maize seedlings roots upon short exposure (10 min to 24 h) to low Cd concentrations (0, 1 or 10 μM): the activity and gene transcript abundance of high-affinity NO(3)(-) transporters, NO(3)(-) reductases and PM H(+)-ATPases were analyzed. Exposure to 1 mM NO(3)(-) led to a peak in high-affinity (0.2 mM) NO(3)(-) uptake rate (induction), which was markedly lowered in Cd-treated roots. Plasma membrane H(+)-ATPase activity was also strongly limited, while internal NO(3)(-) accumulation and NO(3)(-) reductase activity in extracts of Cd treated roots were only slightly lowered. Kinetics of high- and low-affinity NO(3)(-) uptake showed that Cd rapidly (10 min) blocked the inducible high-affinity transport system; the constitutive high-affinity transport system appeared not vulnerable to Cd and the low-affinity transport system appeared to be less affected and only after a prolonged exposure (12 h). Cd-treatment also modified transcript levels of genes encoding high-affinity NO(3)(-) transporters (ZmNTR2.1, ZmNRT2.2), PM H(+)-ATPases (ZmMHA3, ZmMHA4) and NO(3)(-) reductases (ZmNR1, ZmNADH:NR). Despite an expectable increase in NO(3)(-) demand, a negative effect of Cd on NO(3)(-) nutrition is reported. Cd effect results in alterations at the physiological and transcriptional levels of NO(3)(-) uptake from the external solution and it is particularly severe on the inducible high-affinity anion transport system. Furthermore, Cd would limit the capacity of the plant to respond to changes in NO(3) (-) availability.

  15. Sulla carnosa modulates root invertase activity in response to the inhibition of long-distance sucrose transport under magnesium deficiency.

    PubMed

    Farhat, N; Smaoui, A; Maurousset, L; Porcheron, B; Lemoine, R; Abdelly, C; Rabhi, M

    2016-11-01

    Being the principal product of photosynthesis, sucrose is involved in many metabolic processes in plants. As magnesium (Mg) is phloem mobile, an inverse relationship between Mg shortage and sugar accumulation in leaves is often observed. Mg deficiency effects on carbohydrate contents and invertase activities were determined in Sulla carnosa Desf. Plants were grown hydroponically at different Mg concentrations (0.00, 0.01, 0.05 and 1.50 mM Mg) for one month. Mineral analysis showed that Mg contents were drastically diminished in shoots and roots mainly at 0.01 and 0.00 mM Mg. This decline was adversely associated with a significant increase of sucrose, fructose and mainly glucose in shoots of plants exposed to severe deficiency. By contrast, sugar contents were severely reduced in roots of these plants indicating an alteration of carbohydrate partitioning between shoots and roots of Mg-deficient plants. Cell wall invertase activity was highly enhanced in roots of Mg-deficient plants, while the vacuolar invertase activity was reduced at 0.00 mM Mg. This decrease of vacuolar invertase activity may indicate the sensibility of roots to Mg starvation resulting from sucrose transport inhibition. (14) CO2 labeling experiments were in accordance with these findings showing an inhibition of sucrose transport from source leaves to sink tissues (roots) under Mg depletion. The obtained results confirm previous findings about Mg involvement in photosynthate loading into phloem and add new insights into mechanisms evolved by S. carnosa to cope with Mg shortage in particular the increase of the activity of cell wall invertase.

  16. Direct measurement of equilibrium constants for high-affinity hemoglobins.

    PubMed

    Kundu, Suman; Premer, Scott A; Hoy, Julie A; Trent, James T; Hargrove, Mark S

    2003-06-01

    The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) < 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.

  17. The potato sucrose transporter StSUT1 interacts with a DRM-associated protein disulfide isomerase.

    PubMed

    Krügel, Undine; He, Hong-Xia; Gier, Konstanze; Reins, Jana; Chincinska, Izabela; Grimm, Bernhard; Schulze, Waltraud X; Kühn, Christina

    2012-01-01

    Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUT1-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane (DRM) fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiquitin system (SUS) to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane-associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUT1-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was observed between the StSUT1-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUT1-interacting proteins, a protein disulfide isomerase (PDI), interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.

  18. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    PubMed Central

    Alemán, Fernando; Caballero, Fernando; Ródenas, Reyes; Rivero, Rosa M.; Martínez, Vicente; Rubio, Francisco

    2014-01-01

    Potassium (K+) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter. PMID:25228905

  19. High Affinity Iron Permease is Required for Virulence of Rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizopus oryzae is the most common cause of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iro...

  20. High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

    PubMed Central

    Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

    2001-01-01

    In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738

  1. Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars1[W][OA

    PubMed Central

    Fan, Ren-Chun; Peng, Chang-Cao; Xu, Yan-Hong; Wang, Xiao-Fang; Li, Yan; Shang, Yi; Du, Shu-Yuan; Zhao, Rui; Zhang, Xiao-Yan; Zhang, Ling-Yun; Zhang, Da-Peng

    2009-01-01

    Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability. PMID:19502355

  2. Linking Expression of Fructan Active Enzymes, Cell Wall Invertases and Sucrose Transporters with Fructan Profiles in Growing Taproot of Chicory (Cichorium intybus): Impact of Hormonal and Environmental Cues

    PubMed Central

    Wei, Hongbin; Bausewein, Anja; Steininger, Heike; Su, Tao; Zhao, Hongbo; Harms, Karsten; Greiner, Steffen; Rausch, Thomas

    2016-01-01

    In chicory taproot, the inulin-type fructans serve as carbohydrate reserve. Inulin metabolism is mediated by fructan active enzymes (FAZYs): sucrose:sucrose 1-fructosyltransferase (1-SST; fructan synthesis), fructan:fructan-1-fructosyltransferase (1-FFT; fructan synthesis and degradation), and fructan 1-exohydrolases (1-FEH1/2a/2b; fructan degradation). In developing taproot, fructan synthesis is affected by source-to-sink sucrose transport and sink unloading. In the present study, expression of FAZYs, sucrose transporter and CWI isoforms, vacuolar invertase and sucrose synthase was determined in leaf blade, petiole and taproot of young chicory plants (taproot diameter: 2 cm) and compared with taproot fructan profiles for the following scenarios: (i) N-starvation, (ii) abscisic acid (ABA) treatment, (iii) ethylene treatment (via 1-aminoyclopropane-1-carboxylic acid [ACC]), and (iv) cold treatment. Both N-starvation and ABA treatment induced an increase in taproot oligofructans. However, while under N-starvation this increase reflected de novo synthesis, under ABA treatment gene expression profiles indicated a role for both de novo synthesis and degradation of long-chain fructans. Conversely, under ACC and cold treatment oligofructans slightly decreased, correlating with reduced expression of 1-SST and 1-FFT and increased expression of FEHs and VI. Distinct SUT and CWI expression profiles were observed, indicating a functional alignment of SUT and CWI expression with taproot fructan metabolism under different source-sink scenarios. PMID:27994611

  3. His-65 in the proton–sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity

    PubMed Central

    Lu, Jade M.-Y.; Bush, Daniel R.

    1998-01-01

    The proton–sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton–sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction. PMID:9671798

  4. A transgenic study on affecting potato tuber yield by expressing the rice sucrose transporter genes OsSUT5Z and OsSUT2M.

    PubMed

    Sun, Aijun; Dai, Yan; Zhang, Xinsheng; Li, Chunmin; Meng, Kun; Xu, Honglin; Wei, Xiaoli; Xiao, Guifang; Ouwerkerk, Pieter B F; Wang, Mei; Zhu, Zhen

    2011-07-01

    In many plants, sucrose transporters are essential for both sucrose exports from sources and imports into sinks, indicating a function in assimilate partitioning. To investigate whether sucrose transporters can improve the yield of starch plant, potato plants (Solanum tuberosum L. cv. Désirée) were transformed with cDNAs of the rice sucrose transporter genes OsSUT5Z and OsSUT2M under the control of a tuber-specific, class-I patatin promoter. Compared to the controls, the average fructose content of OsSUT5Z transgenic tubers significantly increased. However, the content of the sugars and starch in the OsSUT2M transgenic potato tubers showed no obvious difference. Correspondingly, the average tuber yield, average number of tubers per plant and average weight of single tuber showed no significant difference in OsSUT2M transgenic tubers with controls. In the OsSUT5Z transgenic lines, the average tuber yield per plant was 1.9-fold higher than the controls, and the average number of tubers per plant increased by more than 10 tubers on average, whereas the average weight of a single tuber did not increase significantly. These results suggested that the average number of tubers per plant showed more contribution than the average weight of a single tuber to the tuber yield per plant.

  5. Targeting the AtCWIN1 Gene to Explore the Role of Invertases in Sucrose Transport in Roots and during Botrytis cinerea Infection

    PubMed Central

    Veillet, Florian; Gaillard, Cécile; Coutos-Thévenot, Pierre; La Camera, Sylvain

    2016-01-01

    Cell wall invertases (CWIN) cleave sucrose into glucose and fructose in the apoplast. CWINs are key regulators of carbon partitioning and source/sink relationships during growth, development and under biotic stresses. In this report, we monitored the expression/activity of Arabidopsis cell wall invertases in organs behaving as source, sink, or subjected to a source/sink transition after infection with the necrotrophic fungus Botrytis cinerea. We showed that organs with different source/sink status displayed differential CWIN activities, depending on carbohydrate needs or availabilities in the surrounding environment, through a transcriptional and posttranslational regulation. Loss-of-function mutation of the Arabidopsis cell wall invertase 1 gene, AtCWIN1, showed that the corresponding protein was the main contributor to the apoplastic sucrose cleaving activity in both leaves and roots. The CWIN-deficient mutant cwin1-1 exhibited a reduced capacity to actively take up external sucrose in roots, indicating that this process is mainly dependent on the sucrolytic activity of AtCWIN1. Using T-DNA and CRISPR/Cas9 mutants impaired in hexose transport, we demonstrated that external sucrose is actively absorbed in the form of hexoses by a sugar/H+ symport system involving the coordinated activity of AtCWIN1 with several Sugar Transporter Proteins (STP) of the plasma membrane, i.e., STP1 and STP13. Part of external sucrose was imported without apoplastic cleavage into cwin1-1 seedling roots, highlighting an alternative AtCWIN1-independent pathway for the assimilation of external sucrose. Accordingly, we showed that several genes encoding sucrose transporters of the plasma membrane were expressed. We also detected transcript accumulation of vacuolar invertase (VIN)-encoding genes and high VIN activities. Upon infection, AtCWIN1 was responsible for all the Botrytis-induced apoplastic invertase activity. We detected a transcriptional activation of several AtSUC and AtVIN genes

  6. Targeting the AtCWIN1 Gene to Explore the Role of Invertases in Sucrose Transport in Roots and during Botrytis cinerea Infection.

    PubMed

    Veillet, Florian; Gaillard, Cécile; Coutos-Thévenot, Pierre; La Camera, Sylvain

    2016-01-01

    Cell wall invertases (CWIN) cleave sucrose into glucose and fructose in the apoplast. CWINs are key regulators of carbon partitioning and source/sink relationships during growth, development and under biotic stresses. In this report, we monitored the expression/activity of Arabidopsis cell wall invertases in organs behaving as source, sink, or subjected to a source/sink transition after infection with the necrotrophic fungus Botrytis cinerea. We showed that organs with different source/sink status displayed differential CWIN activities, depending on carbohydrate needs or availabilities in the surrounding environment, through a transcriptional and posttranslational regulation. Loss-of-function mutation of the Arabidopsis cell wall invertase 1 gene, AtCWIN1, showed that the corresponding protein was the main contributor to the apoplastic sucrose cleaving activity in both leaves and roots. The CWIN-deficient mutant cwin1-1 exhibited a reduced capacity to actively take up external sucrose in roots, indicating that this process is mainly dependent on the sucrolytic activity of AtCWIN1. Using T-DNA and CRISPR/Cas9 mutants impaired in hexose transport, we demonstrated that external sucrose is actively absorbed in the form of hexoses by a sugar/H(+) symport system involving the coordinated activity of AtCWIN1 with several Sugar Transporter Proteins (STP) of the plasma membrane, i.e., STP1 and STP13. Part of external sucrose was imported without apoplastic cleavage into cwin1-1 seedling roots, highlighting an alternative AtCWIN1-independent pathway for the assimilation of external sucrose. Accordingly, we showed that several genes encoding sucrose transporters of the plasma membrane were expressed. We also detected transcript accumulation of vacuolar invertase (VIN)-encoding genes and high VIN activities. Upon infection, AtCWIN1 was responsible for all the Botrytis-induced apoplastic invertase activity. We detected a transcriptional activation of several AtSUC and At

  7. Genome-Wide Analysis and Expression Profiling of the SUC and SWEET Gene Families of Sucrose Transporters in Oilseed Rape (Brassica napus L.)

    PubMed Central

    Jian, Hongju; Lu, Kun; Yang, Bo; Wang, Tengyue; Zhang, Li; Zhang, Aoxiang; Wang, Jia; Liu, Liezhao; Qu, Cunmin; Li, Jiana

    2016-01-01

    Sucrose is the principal transported product of photosynthesis from source leaves to sink organs. SUTs/SUCs (sucrose transporters or sucrose carriers) and SWEETs (Sugars Will Eventually be Exported Transporters) play significant central roles in phloem loading and unloading. SUTs/SUCs and SWEETs are key players in sucrose translocation and are associated with crop yields. The SUT/SUC and SWEET genes have been characterized in several plant species, but a comprehensive analysis of these two gene families in oilseed rape has not yet been reported. In our study, 22 and 68 members of the SUT/SUCs and SWEET gene families, respectively, were identified in the oilseed rape (Brassica napus) genome through homology searches. An analysis of the chromosomal distribution, phylogenetic relationships, gene structures, motifs and the cis-acting regulatory elements in the promoters of BnSUC and BnSWEET genes were analyzed. Furthermore, we examined the expression of the 18 BnSUC and 16 BnSWEET genes in different tissues of “ZS11” and the expression of 9 BnSUC and 7 BnSWEET genes in “ZS11” under various conditions, including biotic stress (Sclerotinia sclerotiorum), abiotic stresses (drought, salt and heat), and hormone treatments (abscisic acid, auxin, cytokinin, brassinolide, gibberellin, and salicylic acid). In conclusion, our study provides the first comprehensive analysis of the oilseed rape SUC and SWEET gene families. Information regarding the phylogenetic relationships, gene structure and expression profiles of the SUC and SWEET genes in the different tissues of oilseed rape helps to identify candidates with potential roles in specific developmental processes. Our study advances our understanding of the important roles of sucrose transport in oilseed rape. PMID:27733861

  8. Conformational Changes Represent the Rate-Limiting Step in the Transport Cycle of Maize SUCROSE TRANSPORTER1[C][W

    PubMed Central

    Derrer, Carmen; Wittek, Anke; Bamberg, Ernst; Carpaneto, Armando; Dreyer, Ingo; Geiger, Dietmar

    2013-01-01

    Proton-driven Suc transporters allow phloem cells of higher plants to accumulate Suc to more than 1 M, which is up to ∼1000-fold higher than in the surrounding extracellular space. The carrier protein can accomplish this task only because proton and Suc transport are tightly coupled. This study provides insights into this coupling by resolving the first step in the transport cycle of the Suc transporter SUT1 from maize (Zea mays). Voltage clamp fluorometry measurements combining electrophysiological techniques with fluorescence-based methods enable the visualization of conformational changes of SUT1 expressed in Xenopus laevis oocytes. Using the Suc derivate sucralose, binding of which hinders conformational changes of SUT1, the association of protons to the carrier could be dissected from transport-associated movements of the protein. These combined approaches enabled us to resolve the binding of protons to the carrier and its interrelationship with the alternating movement of the protein. The data indicate that the rate-limiting step of the reaction cycle is determined by the accessibility of the proton binding site. This, in turn, is determined by the conformational change of the SUT1 protein, alternately exposing the binding pockets to the inward and to the outward face of the membrane. PMID:23964025

  9. AmSUT1, a Sucrose Transporter in Collection and Transport Phloem of the Putative Symplastic Phloem Loader Alonsoa meridionalis1

    PubMed Central

    Knop, Christian; Stadler, Ruth; Sauer, Norbert; Lohaus, Gertrud

    2004-01-01

    A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80–91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading. PMID:14730068

  10. Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site.

    PubMed

    Piscitelli, Chayne L; Krishnamurthy, Harini; Gouaux, Eric

    2010-12-23

    Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders, and are the targets of therapeutic and illicit drugs. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

  11. Immunological Evidence for the Existence of a Carrier Protein for Sucrose Transport in Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Root Storage Tissue.

    PubMed Central

    Getz, H. P.; Grosclaude, J.; Kurkdjian, A.; Lelievre, F.; Maretzki, A.; Guern, J.

    1993-01-01

    Monoclonal antibodies were raised in mice against a highly purified tonoplast fraction from isolated red beet (Beta vulgaris L. ssp. conditiva) root vacuoles. Positive hybridoma clones and sub-clones were identified by prescreening using an enzyme-linked immunosorbent assay (ELISA) and by postscreening using a functional assay. This functional assay consisted of testing the impact of hybridoma supernatants and antibody-containing ascites fluids on basal and ATP-stimulated sugar uptake in vacuoles, isolated from protoplasts, as well as in tonoplast vesicles, prepared from tissue homogenates of red beet roots. Antibodies from four clones were particularly positive in ELISAs and they inhibited sucrose uptake significantly. These antibodies were specific inhibitors of sucrose transport, but they exhibited relatively low membrane and species specificity since uptake into red beet root protoplasts and sugarcane tonoplast vesicles was inhibited as well. Fast protein liquid chromatography assisted size exclusion chromatography on Superose 6 columns yielded two major peaks in the 55 to 65-kD regions and in the 110- to 130-kD regions of solubilized proteins from red beet root tonoplasts, which reacted positively in immunoglobulin-M(IgM)-specific ELISAs with anti-sugarcane tonoplast monoclonal IgM antibodies. Only reconstituted proteoliposomes containing polypeptides from the 55- to 65-kD band took up [14C]-sucrose with linear rates for 2 min, suggesting that this fraction contains the tonoplast sucrose carrier. PMID:12231863

  12. Characterization of Sucrose transporter alleles and their association with seed yield-related traits in Brassica napus L

    PubMed Central

    2011-01-01

    Background Sucrose is the primary photosynthesis product and the principal translocating form within higher plants. Sucrose transporters (SUC/SUT) play a critical role in phloem loading and unloading. Photoassimilate transport is a major limiting factor for seed yield. Our previous research demonstrated that SUT co-localizes with yield-related quantitative trait loci. This paper reports the isolation of BnA7.SUT1 alleles and their promoters and their association with yield-related traits. Results Two novel BnA7.SUT1 genes were isolated from B. napus lines 'Eagle' and 'S-1300' and designated as BnA7.SUT1.a and BnA7.SUT1.b, respectively. The BnA7.SUT1 protein exhibited typical SUT features and showed high amino acid homology with related species. Promoters of BnA7.SUT1.a and BnA7.SUT1.b were also isolated and classified as pBnA7.SUT1.a and pBnA7.SUT1.b, respectively. Four dominant sequence-characterized amplified region markers were developed to distinguish BnA7.SUT1.a and BnA7.SUT1.b. The two genes were estimated as alleles with two segregating populations (F2 and BC1) obtained by crossing '3715'×'3769'. BnA7.SUT1 was mapped to the A7 linkage group of the TN doubled haploid population. In silico analysis of 55 segmental BnA7.SUT1 alleles resulted three BnA7.SUT1 clusters: pBnA7.SUT1.a- BnA7.SUT1.a (type I), pBnA7.SUT1.b- BnA7.SUT1.a (type II), and pBnA7.SUT1.b- BnA7.SUT1.b (type III). Association analysis with a diverse panel of 55 rapeseed lines identified single nucleotide polymorphisms (SNPs) in promoter and coding domain sequences of BnA7.SUT1 that were significantly associated with one of three yield-related traits: number of effective first branches (EFB), siliques per plant (SP), and seed weight (n = 1000) (TSW) across all four environments examined. SNPs at other BnA7.SUT1 sites were also significantly associated with at least one of six yield-related traits: EFB, SP, number of seeds per silique, seed yield per plant, block yield, and TSW. Expression levels

  13. New Insights into Phloem Unloading and Expression of Sucrose Transporters in Vegetative Sinks of the Parasitic Plant Phelipanche ramosa L. (Pomel).

    PubMed

    Péron, Thomas; Candat, Adrien; Montiel, Grégory; Veronesi, Christophe; Macherel, David; Delavault, Philippe; Simier, Philippe

    2016-01-01

    The plant-parasitic plant interaction is a interesting model to study sink-source relationship and phloem unloading. The parasitic plants, such as the achlorophyllous plant Phelipanche ramosa, connect to the host phloem through the haustorium and act as supernumerary sinks for the host-derived photoassimilates, primarily sucrose. The application of the fluorescent symplastic tracer, carboxyfluorescein (CF) derived from carboxyfluorescein diacetate (CFDA), to the leaves of the host plant (Brassica napus) showed direct phloem connections at the host-parasite interface. These experiments also evidenced the dominant apoplastic pathway for phloem unloading in major vegetative sinks of the parasite, including tubercles and shoots, except the adventitious root apices. The CF experiments showed also the symplastic isolation of the phloem tissues from the sink tissues in tubercle and shoot of the parasite, then suggesting the pivotal role of sucrose transporters in sucrose unloading in P. ramosa sinks. Three cDNAs encoding sucrose transporters (PrSUT) were isolated from the parasitic plant. PrSUT1 transcripts accumulated at the same level in the tubercle throughout the parasite growth while a significant increase in transcript accumulation occurred after emergence in the flowering shoot, notably in the growing apical part. The in situ hybridization experiments revealed the PrSUT1 transcript accumulation in the mature phloem cells of both subterranean and flowering shoots, as well as in shoot terminal sinks corresponding to apical meristem, scale leaf primordia and immature vasculature. The transient expression experiments in Arabidopsis protoplasts showed that PrSUT1 was localized at the plasma membrane, suggesting its role in phloem functioning and sucrose uptake by the sink cells in P. ramosa. Conversely, the PrSUT2 transcript accumulation was constantly low in tubercles and shoots but PrSUT3 transcripts accumulated markedly in the subterranean and flowering shoots, in

  14. New Insights into Phloem Unloading and Expression of Sucrose Transporters in Vegetative Sinks of the Parasitic Plant Phelipanche ramosa L. (Pomel)

    PubMed Central

    Péron, Thomas; Candat, Adrien; Montiel, Grégory; Veronesi, Christophe; Macherel, David; Delavault, Philippe; Simier, Philippe

    2017-01-01

    The plant-parasitic plant interaction is a interesting model to study sink-source relationship and phloem unloading. The parasitic plants, such as the achlorophyllous plant Phelipanche ramosa, connect to the host phloem through the haustorium and act as supernumerary sinks for the host-derived photoassimilates, primarily sucrose. The application of the fluorescent symplastic tracer, carboxyfluorescein (CF) derived from carboxyfluorescein diacetate (CFDA), to the leaves of the host plant (Brassica napus) showed direct phloem connections at the host-parasite interface. These experiments also evidenced the dominant apoplastic pathway for phloem unloading in major vegetative sinks of the parasite, including tubercles and shoots, except the adventitious root apices. The CF experiments showed also the symplastic isolation of the phloem tissues from the sink tissues in tubercle and shoot of the parasite, then suggesting the pivotal role of sucrose transporters in sucrose unloading in P. ramosa sinks. Three cDNAs encoding sucrose transporters (PrSUT) were isolated from the parasitic plant. PrSUT1 transcripts accumulated at the same level in the tubercle throughout the parasite growth while a significant increase in transcript accumulation occurred after emergence in the flowering shoot, notably in the growing apical part. The in situ hybridization experiments revealed the PrSUT1 transcript accumulation in the mature phloem cells of both subterranean and flowering shoots, as well as in shoot terminal sinks corresponding to apical meristem, scale leaf primordia and immature vasculature. The transient expression experiments in Arabidopsis protoplasts showed that PrSUT1 was localized at the plasma membrane, suggesting its role in phloem functioning and sucrose uptake by the sink cells in P. ramosa. Conversely, the PrSUT2 transcript accumulation was constantly low in tubercles and shoots but PrSUT3 transcripts accumulated markedly in the subterranean and flowering shoots, in

  15. Tissue-specific transcriptome analysis within the maturing sugarcane stalk reveals spatial regulation in the expression of cellulose synthase and sucrose transporter gene families.

    PubMed

    Casu, Rosanne E; Rae, Anne L; Nielsen, Janine M; Perroux, Jai M; Bonnett, Graham D; Manners, John M

    2015-12-01

    Sugarcane (Saccharum spp. hybrids) accumulates high concentrations of sucrose in its mature stalk and a considerable portion of carbohydrate metabolism is also devoted to cell wall synthesis and fibre production. We examined tissue-specific expression patterns to explore the spatial deployment of pathways responsible for sucrose accumulation and fibre synthesis within the stalk. We performed expression profiling of storage parenchyma, vascular bundles and rind dissected from a maturing stalk internode of sugarcane, identifying ten cellulose synthase subunit genes and examining significant differences in the expression of their corresponding transcripts and those of several sugar transporters. These were correlated with differential expression patterns for transcripts of genes encoding COBRA-like proteins and other cell wall metabolism-related proteins. The sugar transporters genes ShPST2a, ShPST2b and ShSUT4 were significantly up-regulated in storage parenchyma while ShSUT1 was up-regulated in vascular bundles. Two co-ordinately expressed groups of cell wall related transcripts were also identified. One group, associated with primary cell wall synthesis (ShCesA1, ShCesA7, ShCesA9 and Shbk2l3), was up-regulated in parenchyma. The other group, associated with secondary cell wall synthesis (ShCesA10, ShCesA11, ShCesA12 and Shbk-2), was up-regulated in rind. In transformed sugarcane plants, the ShCesA7 promoter conferred stable expression of green fluorescent protein preferentially in the storage parenchyma of the maturing stalk internode. Our results indicate that there is spatial separation for elevated expression of these important targets in both sucrose accumulation and cell wall synthesis, allowing for increased clarity in our understanding of sucrose transport and fibre synthesis in sugarcane.

  16. Radiosynthesis of 6’-Deoxy-6’[18F]Fluorosucrose via Automated Synthesis and Its Utility to Study In Vivo Sucrose Transport in Maize (Zea mays) Leaves

    PubMed Central

    Ying, Weijiang; Gaddam, Vikram; Harmata, Michael; Robertson, J. David; Swyers, Michael; Jurisson, Silvia S.

    2015-01-01

    Sugars produced from photosynthesis in leaves are transported through the phloem tissues within veins and delivered to non-photosynthetic organs, such as roots, stems, flowers, and seeds, to support their growth and/or storage of carbohydrates. However, because the phloem is located internally within the veins, it is difficult to access and to study the dynamics of sugar transport. Radioactive tracers have been extensively used to study vascular transport in plants and have provided great insights into transport dynamics. To better study sucrose partitioning in vivo, a novel radioactive analog of sucrose was synthesized through a completely chemical synthesis route by substituting fluorine-18 (half-life 110 min) at the 6’ position to generate 6’-deoxy-6’[18F]fluorosucrose (18FS). This radiotracer was then used to compare sucrose transport between wild-type maize plants and mutant plants lacking the Sucrose transporter1 (Sut1) gene, which has been shown to function in sucrose phloem loading. Our results demonstrate that 18FS is transported in vivo, with the wild-type plants showing a greater rate of transport down the leaf blade than the sut1 mutant plants. A similar transport pattern was also observed for universally labeled [U-14C]sucrose ([U-14C]suc). Our findings support the proposed sucrose phloem loading function of the Sut1 gene in maize, and additionally demonstrate that the 18FS analog is a valuable, new tool that offers imaging advantages over [U-14C]suc for studying phloem transport in plants. PMID:26024520

  17. The Control of Single-Celled Cotton Fiber Elongation by Developmentally Reversible Gating of Plasmodesmata and Coordinated Expression of Sucrose and K+ Transporters and Expansin

    PubMed Central

    Ruan, Yong-Ling; Llewellyn, Danny J.; Furbank, Robert T.

    2001-01-01

    Each cotton fiber is a single cell that elongates to 2.5 to 3.0 cm from the seed coat epidermis within ∼16 days after anthesis (DAA). To elucidate the mechanisms controlling this rapid elongation, we studied the gating of fiber plasmodesmata and the expression of the cell wall–loosening gene expansin and plasma membrane transporters for sucrose and K+, the major osmotic solutes imported into fibers. Confocal imaging of the membrane-impermeant fluorescent solute carboxyfluorescein (CF) revealed that the fiber plasmodesmata were initially permeable to CF (0 to 9 DAA), but closed at ∼10 DAA and re-opened at 16 DAA. A developmental switch from simple to branched plasmodesmata was also observed in fibers at 10 DAA. Coincident with the transient closure of the plasmodesmata, the sucrose and K+ transporter genes were expressed maximally in fibers at 10 DAA with sucrose transporter proteins predominately localized at the fiber base. Consequently, fiber osmotic and turgor potentials were elevated, driving the rapid phase of elongation. The level of expansin mRNA, however, was high at the early phase of elongation (6 to 8 DAA) and decreased rapidly afterwards. The fiber turgor was similar to the underlying seed coat cells at 6 to 10 DAA and after 16 DAA. These results suggest that fiber elongation is initially achieved largely by cell wall loosening and finally terminated by increased wall rigidity and loss of higher turgor. To our knowledge, this study provides an unprecedented demonstration that the gating of plasmodesmata in a given cell is developmentally reversible and is coordinated with the expression of solute transporters and the cell wall–loosening gene. This integration of plasmodesmatal gating and gene expression appears to control fiber cell elongation. PMID:11158528

  18. Structural origins of high-affinity biotin binding to streptavidin.

    PubMed

    Weber, P C; Ohlendorf, D H; Wendoloski, J J; Salemme, F R

    1989-01-06

    The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative deformations in the twisted beta sheets that link tetramer subunits.

  19. High affinity choline uptake: an early index of cholinergic innervation in rat brain.

    PubMed

    Sorimachi, M; Kataoka, K

    1975-08-29

    The uptake of [3H]choline was investigated in nuclei-free homogenates or crude synaptosomal fractions (P2) from rat brain under various stages of development. A comparable sensitivity of uptake to treatment by hyposmotic shock suggested the involvement of synaptosomal populations in choline uptake in immature as well as in adult brains. However, significant changes in the "apparent" Km for the high affinity transport system and quantitative differences in the Na ion requirement for maximal uptake at 0.43 muM choline concentration were found during development; facts which suggested a greater contribution of the low affinity system in the more immature brains. Assuming that the uptake with high and low sensitivity to Na+ reduction reflected that via the high and low affinity system reslectively, we have attempted to obtain real Km values for the high affinity system. These Km values changed less than those measured directly, suggesting that the affinity constant for the high affinity system does not change during development. On these assumptions, the developmental changes of cholinergic synaptogenesis were examined in 5 distinct regions of the brain. It was found that the synaptogenesis begins several days earlier than the increase of choline acetyltransferase (ChAc) level in the frontal cortex, the hippocampus, the superior colliculus and the cerebellum. These regions may be included among the terminal-rich regions according to available evidence related to cholinergic systems. On the other hand, synaptogenesis accompanied the concomitant ChAc increase in the striatum, where the cholinergic interneurons are present. It is concluded that the increase of ChAc in the terminal-rich regions is delayed by the axoplasmic flow; therefore, the earlier index of cholinergic synaptogenesis in these regions is the high affinity uptake activity rather than the enzyme activity.

  20. Selective high affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  1. Ethylene stimulation of latex yield depends on the expression of a sucrose transporter (HbSUT1B) in rubber tree (Hevea brasiliensis).

    PubMed

    Dusotoit-Coucaud, Anaïs; Kongsawadworakul, Panida; Maurousset, Laurence; Viboonjun, Unshira; Brunel, Nicole; Pujade-Renaud, Valérie; Chrestin, Hervé; Sakr, Soulaïman

    2010-12-01

    Hevea brasiliensis is an important industrial crop for natural rubber production. Latex biosynthesis occurs in the cytoplasm of highly specialized latex cells and requires sucrose as the unique precursor. Ethylene stimulation of latex production results in high sugar flow from the surrounding cells of inner bark towards the latex cells. The aim of this work was to understand the role of seven sucrose transporters (HbSUTs) and one hexose transporter (HbHXT1) in this process. Two Hevea clones were used: PB217 and PB260, respectively described as high and low yielding clones. The expression pattern of these sugar transporters (HbSUTs and HbHXT1) was monitored under different physiological conditions and found to be maximal in latex cells. HbSUT1, one of the most abundant isoforms, displayed the greatest response to ethylene treatment. In clone PB217, ethylene treatment led to a higher accumulation of HbSUT1B in latex cells than in the inner bark tissues. Conversely, stronger expression of HbSUT1B was observed in inner bark tissues than in latex cells of PB260. A positive correlation with HbSUT1B transcript accumulation and increased latex production was further supported by its lower expression in latex cells of the virgin clone PB217.

  2. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

    SciTech Connect

    Matt Vaughn Greg Harrington Daniel R Bush

    2002-08-06

    This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with the loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose

  3. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    SciTech Connect

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  4. The Plasma Membrane-Localized Sucrose Transporter IbSWEET10 Contributes to the Resistance of Sweet Potato to Fusarium oxysporum

    PubMed Central

    Li, Yan; Wang, Yannan; Zhang, Huan; Zhang, Qian; Zhai, Hong; Liu, Qingchang; He, Shaozhen

    2017-01-01

    SWEET (Sugars Will Eventually be Exported Transporter) proteins, a novel family of sugar transporters, mediate the diffusion of sugars across cell membranes and acts as key players in sucrose phloem loading. Manipulation of SWEET genes in plants leads to various effects on resistance to biotic and abiotic stresses due to disruption of sugar efflux and changes in sugar distribution. In this study, a member of the SWEET gene family, IbSWEET10, was cloned from the sweet potato line ND98. mRNA expression analysis in sweet potato and promoter β-Glucuronidase analysis in Arabidopsis showed that IbSWEET10 is highly expressed in leaves, especially in vascular tissue. Transient expression in tobacco epidermal cells revealed plasma membrane localization of IbSWEET10, and heterologous expression assays in yeast indicated that IbSWEET10 encodes a sucrose transporter. The expression level of IbSWEET10 was significantly up-regulated in sweet potato infected with Fusarium oxysporum Schlecht. f. sp. batatas. Further characterization revealed IbSWEET10-overexpressing sweet potato lines to be more resistant to F. oxysporum, exhibiting better growth after infection compared with the control; conversely, RNA interference (RNAi) lines showed the opposite results. Additionally, the sugar content of IbSWEET10-overexpression sweet potato was significantly reduced, whereas that in RNAi plants was significantly increased compared with the control. Therefore, we suggest that the reduction in sugar content caused by IbSWEET10 overexpression is the major reason for the enhanced F. oxysporum resistance of the transgenic plants. This is the first report that the IbSWEET10 transporter contributes to the resistance of sweet potato to F. oxysporum. The IbSWEET10 gene has the great potential to be used for improving the resistance to F. oxysporum in sweet potato and other plants. PMID:28261250

  5. Rice sucrose transporter1 (OsSUT1) up-regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles.

    PubMed

    Ibraheem, O; Botha, C E J; Bradley, G; Dealtry, G; Roux, S

    2014-07-01

    The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3-week-old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1- to 10-day infestation time course, using a combination of gene expression and β-glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide-field fluorescence microscopy was used to confirm the expression of OsSUT1-promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up-regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up-regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid-related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves.

  6. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  7. HIGH AFFINITY, DSRNA BINDING BY DISCONNECTED INTERACTING PROTEIN 1†

    PubMed Central

    Catanese, Daniel J.; Matthews, Kathleen S.

    2010-01-01

    Disconnected Interacting Protein 1 (DIP1) appears from sequence analysis and preliminary binding studies to be a member of the dsRNA-binding protein family. Of interest, DIP1 was shown previously to interact with and influence multiple proteins involved in transcription regulation in Drosophila melanogaster. We show here that the longest isoform of this protein, DIP1-c, exhibits a 500-fold preference for dsRNA over dsDNA of similar nucleotide sequence. Further, DIP1-c demonstrated very high affinity for a subset of dsRNA ligands, with binding in the picomolar range for VA1 RNA and miR-iab-4 precursor stem-loop, a potential physiological RNA target involved in regulating expression of its protein partner, Ultrabithorax. PMID:20643095

  8. Transport of sucrose-modified nanoscale zero-valent iron in saturated porous media: role of media size, injection rate and input concentration.

    PubMed

    Li, Hui; Zhao, Yong-sheng; Han, Zhan-tao; Hong, Mei

    2015-01-01

    The growing use of nanoscale zero-valent iron (NZVI) in the remediation of contaminated groundwater raises concerns regarding its transport in aquifers. Laboratory-scale sand-packed column experiments were conducted with bare and sucrose-modified NZVI (SM-NZVI) to improve our understanding of the transport of the nanoparticles in saturated porous media, as well as the role of media size, suspension injection rate and concentration on the nanoparticle behavior. As the main indicative parameters, the normalized effluent concentration was measured and the deposition rate coefficient (k) was calculated for different simulated conditions. Overall, compared to the high retention of bare NZVI in the saturated silica column, SM-NZVI suspension could travel through the coarse sand column easily. However, the transport of SM-NZVI particles was not very satisfactory in a smaller size granular matrix especially in fine silica sand. Furthermore, the value of k regularly decreased with the increasing injection rate of suspension but increased with suspension concentration, which could reflect the role of these factors in the SM-NZVI travel process. The calculation of k-value at the tests condition adequately described the experimental results from the point of deposition dynamics, which meant the assumption of first-order deposition kinetics for the transport of NZVI particles was reasonable and feasible.

  9. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium

    PubMed Central

    1994-01-01

    Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin- minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine- coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development. PMID:8089176

  10. Phosphatidylserine Reversibly Binds Cu2+ with Extremely High Affinity

    PubMed Central

    Monson, Christopher F.; Cong, Xiao; Robison, Aaron; Pace, Hudson P.; Liu, Chunming; Poyton, Matthew F.; Cremer, Paul S.

    2012-01-01

    Phosphatidylserine (PS) embedded within supported lipid bilayers (SLBs) was found to bind Cu2+ from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu2+ to PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85–90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu2+ concentrations and basic values pH (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion limited complex formation at basic pH, but rapid dissociation under acidic conditions. The tight binding of Cu2+ to PS may have physiological consequences under certain circumstances. PMID:22548290

  11. Reducing sucrose loss in sugarbeet storage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose loss in sugarbeet storage is a considerable problem which is negatively influenced by environmental conditions, stress on roots from disease problems in the field, rough handling during harvest and transport, and microbial growth. To reduce sucrose loss in sugarbeet storage, studies were in...

  12. Water Deficit Enhances C Export to the Roots in Arabidopsis thaliana Plants with Contribution of Sucrose Transporters in Both Shoot and Roots1[OPEN

    PubMed Central

    Durand, Mickaël; Porcheron, Benoît; Maurousset, Laurence; Lemoine, Rémi; Pourtau, Nathalie

    2016-01-01

    Root high plasticity is an adaptation to its changing environment. Water deficit impairs growth, leading to sugar accumulation in leaves, part of which could be available to roots via sucrose (Suc) phloem transport. Phloem loading is widely described in Arabidopsis (Arabidopsis thaliana), while unloading in roots is less understood. To gain information on leaf-to-root transport, a soil-based culture system was developed to monitor root system architecture in two dimensions. Under water deficit (50% of soil water-holding capacity), total root length was strongly reduced but the depth of root foraging and the shape of the root system were less affected, likely to improve water uptake. 14CO2 pulse-chase experiments confirmed that water deficit enhanced carbon (C) export to the roots, as suggested by the increased root-to-shoot ratio. The transcript levels of AtSWEET11 (for sugar will eventually be exported transporter), AtSWEET12, and AtSUC2 (for Suc carrier) genes, all three involved in Suc phloem loading, were significantly up-regulated in leaves of water deficit plants, in accordance with the increase in C export from the leaves to the roots. Interestingly, the transcript levels of AtSUC2 and AtSWEET11 to AtSWEET15 were also significantly higher in stressed roots, underlying the importance of Suc apoplastic unloading in Arabidopsis roots and a putative role for these Suc transporters in Suc unloading. These data demonstrate that, during water deficit, plants respond to growth limitation by allocating relatively more C to the roots to maintain an efficient root system and that a subset of Suc transporters is potentially involved in the flux of C to and in the roots. PMID:26802041

  13. Sucrose preload reduces snacking after mild mental stress in healthy participants as a function of 5-hydroxytryptamine transporter gene promoter polymorphism.

    PubMed

    Markus, C Rob; Jonkman, Lisa M; Capello, Aimee; Leinders, Sacha; Hüsch, Fabian

    2015-01-01

    Brain serotonin (5-hydroxytryptamine, 5-HT) dysfunction is considered to promote food intake and eating-related disturbances, especially under stress or negative mood. Vulnerability for 5-HT disturbances is considered to be genetically determined, including a short (S) allele polymorphism in the serotonin transporter gene (5-HTTLPR) that is associated with lower serotonin function. Since 5-HT function may be slightly increased by carbohydrate consumption, S-allele 5-HTTLPR carriers in particular may benefit from a sugar-preload due to their enhanced 5-HT vulnerability. The aim of the current study was to investigate whether a sugar-containing preload may reduce appetite and energy intake after exposure to stress to induce negative mood, depending on genetic 5-HT vulnerability. From a population of 771 healthy young male and female genotyped college students 31 S/S carriers (8 males, 23 females) and 26 long allele (L/L) carriers (9 males, 17 females) (mean ± S.D. 22 ± 1.6 years; body mass index, BMI, 18-33 kg/m(2)) were monitored for changes in appetite and snacking behavior after stress exposure. Results revealed an increased energy intake after mild mental stress (negative mood) mainly for high-fat sweet foods, which was significantly greater in S/S carriers, and only in these genotypes this intake was significantly reduced by a sucrose-containing preload. Although alternative explanations are possible, it is suggested that S/S participants may have enhanced brain (hypothalamic) 5-HT responsiveness to food that makes them more susceptible to the beneficial satiation effects of a sucrose-preload as well as to the negative effects of mild mental stress on weight gain.

  14. High affinity binding of [3H]-tyramine in the central nervous system.

    PubMed Central

    Vaccari, A.

    1986-01-01

    Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine. PMID:3801770

  15. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  16. A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds.

    PubMed

    Weber, H; Borisjuk, L; Heim, U; Sauer, N; Wobus, U

    1997-06-01

    To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUT1 and VfSTP1, respectively. sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUT1 and VfSTP1 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTP1 mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUT1 transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT1 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability.

  17. Characterization of AMT-mediated high-affinity ammonium uptake in roots of maize (Zea mays L.).

    PubMed

    Gu, Riliang; Duan, Fengying; An, Xia; Zhang, Fusuo; von Wirén, Nicolaus; Yuan, Lixing

    2013-09-01

    High-affinity ammonium uptake in plant roots is mainly mediated by AMT1-type ammonium transporters, and their regulation varies depending on the plant species. In this study we aimed at characterizing AMT-mediated ammonium transport in maize, for which ammonium-based fertilizer is an important nitrogen (N) source. Two ammonium transporter genes, ZmAMT1;1a and ZmAMT1;3, were isolated from a maize root-specific cDNA library by functional complementation of an ammonium uptake-defective yeast mutant. Ectopic expression of both genes in an ammonium uptake-defective Arabidopsis mutant conferred high-affinity ammonium uptake capacities in roots with substrate affinities of 48 and 33 μM for ZmAMT1;1a and ZmAMT1;3, respectively. In situ hybridization revealed co-localization of both ZmAMT genes on the rhizodermis, suggesting an involvement in capturing ammonium from the rhizosphere. In N-deficient maize roots, influx increased significantly while ZmAMT expression did not. Ammonium resupply to N-deficient or nitrate-pre-cultured roots, however, rapidly enhanced both influx and ZmAMT transcript levels, revealing a substrate-inducible regulation of ammonium uptake. In conclusion, the two rhizodermis-localized transporters ZmAMT1;1a and ZmAMT1;3 are most probably the major components in the high-affinity transport system in maize roots. A particular regulatory feature is their persistent induction by ammonium rather than an up-regulation under N deficiency.

  18. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  19. Iron Sucrose Injection

    MedlinePlus

    Iron sucrose injection is used treat iron-deficiency anemia (a lower than normal number of red blood cells due ... and may cause the kidneys to stop working). Iron sucrose injection is in a class of medications called iron ...

  20. Sucrose and Related Oligosaccharides

    NASA Astrophysics Data System (ADS)

    Eggleston, Gillian

    Sucrose (α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside) is the most common low-molecular-weight sugar found in the plant kingdom. It is ubiquitously known as common table sugar and primarily produced industrially from sugarcane (Saccharum officinarum) and sugar beet (Beta vulgaris); the basics of the industrial manufacture of sucrose are outlined in this chapter. Commercial sucrose has a very high purity (> 99.9%) making it one of the purest organic substances produced on an industrial scale. Value-addition to sucrose via chemical and biotechnological reactions is becoming more important for the diversification of the sugar industry to maintain the industries' competitiveness in a world increasingly turning to a bio-based economy. The basis for the chemical reactivity of sucrose is the eight hydroxyl groups present on the molecule, although, sucrose chemical reactivity is regarded as difficult. Increasing use of enzymatic biotechnological techniques to derivatize sucrose is expected, to add special functionalities to sucrose products like biodegradability, biocompatibility, and non-toxicity. Analysis of sucrose by colorimetric, enzymatic, oxidation-reduction and chromatography methods are discussed. Oligosaccharides related to sucrose are outlined in detail and include sucrose-based plant, honey and in vitro oligosaccharides.

  1. Stable U(IV) complexes form at high-affinity mineral surface sites.

    PubMed

    Latta, Drew E; Mishra, Bhoopesh; Cook, Russell E; Kemner, Kenneth M; Boyanov, Maxim I

    2014-01-01

    Uranium (U) poses a significant contamination hazard to soils, sediments, and groundwater due to its extensive use for energy production. Despite advances in modeling the risks of this toxic and radioactive element, lack of information about the mechanisms controlling U transport hinders further improvements, particularly in reducing environments where U(IV) predominates. Here we establish that mineral surfaces can stabilize the majority of U as adsorbed U(IV) species following reduction of U(VI). Using X-ray absorption spectroscopy and electron imaging analysis, we find that at low surface loading, U(IV) forms inner-sphere complexes with two metal oxides, TiO2 (rutile) and Fe3O4 (magnetite) (at <1.3 U nm(-2) and <0.037 U nm(-2), respectively). The uraninite (UO2) form of U(IV) predominates only at higher surface loading. U(IV)-TiO2 complexes remain stable for at least 12 months, and U(IV)-Fe3O4 complexes remain stable for at least 4 months, under anoxic conditions. Adsorbed U(IV) results from U(VI) reduction by Fe(II) or by the reduced electron shuttle AH2QDS, suggesting that both abiotic and biotic reduction pathways can produce stable U(IV)-mineral complexes in the subsurface. The observed control of high-affinity mineral surface sites on U(IV) speciation helps explain the presence of nonuraninite U(IV) in sediments and has important implications for U transport modeling.

  2. Succinic acid production from sucrose by Actinobacillus succinogenes NJ113.

    PubMed

    Jiang, Min; Dai, Wenyu; Xi, Yonglan; Wu, Mingke; Kong, Xiangping; Ma, Jiangfeng; Zhang, Min; Chen, Kequan; Wei, Ping

    2014-02-01

    In this study, sucrose, a reproducible disaccharide extracted from plants, was used as the carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. During serum bottle fermentation, the succinic acid concentration reached 57.1g/L with a yield of 71.5%. Further analysis of the sucrose utilization pathways revealed that sucrose was transported and utilized via a sucrose phosphotransferase system, sucrose-6-phosphate hydrolase, and a fructose PTS. Compared to glucose utilization in single pathway, more pathways of A. succinogenes NJ113 are dependent on sucrose utilization. By changing the control strategy in a fed-batch culture to alleviate sucrose inhibition, 60.5g/L of succinic acid was accumulated with a yield of 82.9%, and the productivity increased by 35.2%, reaching 2.16g/L/h. Thus utilization of sucrose has considerable potential economics and environmental meaning.

  3. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  4. In vivo gene transfer to dopamine neurons of rat substantia nigra via the high-affinity neurotensin receptor.

    PubMed Central

    Alvarez-Maya, I.; Navarro-Quiroga, I.; Meraz-Ríos, M. A.; Aceves, J.; Martinez-Fong, D.

    2001-01-01

    BACKGROUND: Recently, we synthesized a nonviral gene vector capable of transfecting cell lines taking advantage of neurotensin (NT) internalization. The vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polyplex. This hypothesis was tested here using reporter genes encoding green fluorescent protein or chloramphenicol acetyl transferase. MATERIALS AND METHODS: NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involved in the propidium iodide-labeled polyplex internalization and reporter gene expression. RESULTS: Polyplex internalization was observed within dopamine neurons but not within glial cells, and was prevented by both hypertonic sucrose solution and SR-48692, a selective nonpeptide antagonist of NT receptors. Reporter gene expression was observed in dopamine neurons from 48 hr up to 15 days after NT-polyplex injection, and was prevented by SR-48692. However, no expression was seen when the NT-polyplex was injected into the ansiform lobule of the cerebellum, which contains low- but not high-affinity NT receptors. Neither internalization nor expression was observed in cultured glial cells, despite the NT-polyplex binding to those cells that was prevented by levocabastine, a low-affinity NT receptor antagonist. CONCLUSIONS: These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in vivo. NT polyfection may be used to transfer genes of physiologic interest to nigrostriatal dopamine neurons, and to produce transgenic animal models of dopamine-related diseases. PMID:11471555

  5. Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves: estimation by alternate substrate utilization

    SciTech Connect

    Schmalstig, J.G.; Hitz, W.D.

    1987-10-01

    The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1'-deoxy-1'-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS.(/sup 14/C)Sucrose and (/sup 14/C)FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when (/sup 14/C)sucrose was supplied than when (/sup 14/C)FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%.

  6. Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

    PubMed Central

    Silva-Bailão, Mirelle Garcia; Bailão, Elisa Flávia Luiz Cardoso; Lechner, Beatrix Elisabeth; Gauthier, Gregory M.; Lindner, Herbert; Bailão, Alexandre Melo; Haas, Hubertus; de Almeida Soares, Célia Maria

    2014-01-01

    Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity. PMID:25157575

  7. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  8. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  9. Regulation of high affinity iron uptake in the yeast Saccharomyces cerevisiae. Role of dioxygen and Fe.

    PubMed

    Hassett, R F; Romeo, A M; Kosman, D J

    1998-03-27

    High affinity iron uptake in Saccharomyces cerevisiae requires a metal reductase, a multicopper ferroxidase, and an iron permease. Fet3, the apparent ferroxidase, is proposed to facilitate iron uptake by catalyzing the oxidation of reductase-generated Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1 (Kaplan, J., and O'Halloran, T. V. (1996) Science 271, 1510-1512). We show here that dioxygen also plays an essential role in the expression of these iron uptake activities. Cells grown anaerobically exhibited no Fe(III) reductase or high affinity iron uptake activity, even if assayed for these activities under air. Northern blot analysis showed that the amount of those mRNAs encoding proteins associated with this uptake was repressed in anaerobic cultures but was rapidly induced by exposure of the culture to dioxygen. The anaerobic repression was reduced in cells expressing an iron-independent form of the trans-activator, Aft1, a protein that regulates the expression of these proteins. Thus, the effect of oxygenation on this expression appeared due at least in part to the state or distribution of iron in the cells. In support of this hypothesis, the membrane-permeant Fe(II) chelator, 2, 2'-bipyridyl, in contrast to the impermeant chelator bathophenanthroline disulfonate, caused a strong and rapid induction of these transcripts under anaerobic conditions. An increase in the steady-state levels of iron-regulated transcripts upon oxygenation or 2,2'-bipyridyl addition occurred within 5 min, indicating that a relatively small, labile intracellular pool of Fe(II) regulates the expression of these activities. The strength of the anaerobic repression was dependent on the low affinity, Fe(II)-specific iron transporter, encoded by FET4, suggesting that this Fe(II) pool was linked in part to iron brought into the cell via Fet4 protein. The data suggest a model in which dioxygen directly or indirectly modulates the Fe

  10. Sucrose signaling in plants

    PubMed Central

    Tognetti, Jorge A.; Pontis, Horacio G.; Martínez-Noël, Giselle M.A.

    2013-01-01

    The role of sucrose as a signaling molecule in plants was originally proposed several decades ago. However, recognition of sucrose as a true signal has been largely debated and only recently this role has been fully accepted. The best-studied cases of sucrose signaling involve metabolic processes, such as the induction of fructan or anthocyanin synthesis, but a large volume of scattered information suggests that sucrose signals may control a vast array of developmental processes along the whole life cycle of the plant. Also, wide gaps exist in our current understanding of the intracellular steps that mediate sucrose action. Sucrose concentration in plant tissues tends to be directly related to light intensity, and inversely related to temperature, and accordingly, exogenous sucrose supply often mimics the effect of high light and cold. However, many exceptions to this rule seem to occur due to interactions with other signaling pathways. In conclusion, the sucrose role as a signal molecule in plants is starting to be unveiled and much research is still needed to have a complete map of its significance in plant function. PMID:23333971

  11. ( sup 14 C)-Sucrose uptake by guard cell protoplasts of pisum sativum, argenteum mutant

    SciTech Connect

    Rohrig, K.; Raschke, K. )

    1991-05-01

    Guard cells rely on import for their supply with reduced carbon. The authors tested by silicone oil centrifugation the ability of guard cell protoplasts to accumulated ({sup 14}C)-sucrose. Uptake rates were corrected after measurement of {sup 14}C-sorbitol and {sup 3}H{sub 2}O spaces. Sucrose uptake followed biphasic kinetics, with a high-affinity component below 1 mM external sucrose (apparent K{sub m} 0.8 mM at 25C) and a low-affinity nonsaturable component above. Uptake depended on pH (optimum at pH 5.0). Variations in the concentrations of external KCl, CCCP, and valinomycin indicated that about one-half of the sucrose uptake rate could be related to an electrochemical gradient across the plasmalemma. Total uptake rates measured at 5 mM external sucrose seem to be sufficient to replenish emptied plastids with starch within a few hours.

  12. Sucrose and behavioral problems.

    PubMed

    Benton, David

    2008-05-01

    Various mechanisms by which sucrose could influence behavior are reviewed. Firstly there is food intolerance. There are dozens of foods to which an adverse reaction has been demonstrated, although a reaction to sucrose is less frequent than many other foods. A second possible mechanism is hypoglycemia. There is evidence that a tendency to develop low blood glucose levels, but higher than those that can be described clinically as hypoglycemic, is associated with irritability and violence. However, sucrose is not the predominant cause of swings in blood glucose levels. Thirdly, the role of sucrose intake on micro-nutrient status has been considered as studies have found that micro-nutrient supplementation decreased anti-social behavior. Micro-nutrient intake is more closely associated with the total energy rather than sucrose intake; typically the amount of sucrose in the diet does not lead to micro-nutrient deficiency. In fact meta-analysis of well designed studies that have examined the impact of sucrose on the behavior of children produced no evidence that it has an adverse influence.

  13. MKC-231, a choline uptake enhancer: (3) Mode of action of MKC-231 in the enhancement of high-affinity choline uptake.

    PubMed

    Takashina, Ken; Bessho, Tomoko; Mori, Reiko; Kawai, Kunji; Eguchi, Junichi; Saito, Ken-Ichi

    2008-07-01

    MKC-231, a putative cholinergic activity, is reported to improve learning and memory impaired in AF64A-treated animals. MKC-231 enhances high-affinity choline uptake (HACU) known as the rate-limiting step of acetylcholine (ACh) synthesis. We investigated the mode of action (MOA) of HACU enhancement by MKC-231. Intracerebroventricular (i.c.v.) injections of AF64A (3 nmol/brain) resulted in significant HACU reduction in hippocampal synaptosomes. Treatment with MKC-231 increased Vmax of HACU and Bmax of [3H]-HC-3 binding 1.6 and 1.7-fold, respectively. In studies of [3H]-MKC-231 binding and Biacore analysis, MKC-231 showed noticeable affinity for cloned high-affinity choline transporters (CHT1). The present study suggests that MKC-231 directly affects trafficking of CHT1 and increases the numbers of transporter, working for HACU, at the synaptic membrane.

  14. The shoot is important for high-affinity nitrate uptake in Egeria densa, a submerged vascular plant.

    PubMed

    Takayanagi, Shu; Takagi, Yuma; Shimizu, Akifumi; Hasegawa, Hiroshi

    2012-09-01

    To understand the mechanisms of nitrate uptake by submerged vascular plants, a cDNA for a high-affinity nitrate transporter, NRT2, was isolated from Egeria densa, a submerged monocot. The deduced EdNRT2 protein was similar to the proteins of a conserved NRT2 group in higher plants. Real-time reverse transcription-PCR analysis revealed that after feeding whole plants with 0.2 mM nitrate, the EdNRT2 transcripts were induced in both shoots and roots within 0.5 h, reached the maximum by 1-3 h and then decreased. The EdNRT2 transcript levels in shoots were comparable to those in roots. When nitrate was applied separately to shoots and roots, the EdNRT2 transcripts were induced only in nitrate-treated organs and reached the maximum levels comparable to those in organs when nitrate was applied to whole plants. (15)N-nitrate feeding experiments demonstrated that both shoots and roots are responsible for nitrate uptake and that biomass and (15)N content in shoots was even higher than that in roots. We concluded that EdNRT2 is involved in high-affinity nitrate uptake by shoots and roots of E. densa, that nitrate is taken up independently by shoots and roots and that shoots play an important role in nitrate uptake from aquatic ecosystem.

  15. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    PubMed

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  16. Sertraline and its metabolite desmethylsertraline, but not bupropion or its three major metabolites, have high affinity for P-glycoprotein.

    PubMed

    Wang, Jun-Sheng; Zhu, Hao-Jie; Gibson, Bryan Bradford; Markowitz, John Seth; Donovan, Jennifer Lyn; DeVane, Carl Lindsay

    2008-02-01

    The ATP-binding cassette (ABC) transporter protein subfamily B1 line (ABCB1) transporter P-glycoprotein (P-gp) plays an important role in the blood-brain barrier limiting a broad spectrum of substrates from entering the central nervous system. In the present study, the transport activity of P-gp for sertraline, desmethylsertraline, bupropion, and the major metabolites of bupropion, threo-amino alcohol (TB), erythro-amino alcohol (EB), and hydroxy metabolite (HB) was studied using an ATPase assay in expressed human P-gp membranes by measuring concentrations of inorganic P(i) in expressed human P-gp membranes. Verapamil was included as a positive control. The Michaelis-Menten equation was used for characterizing the kinetic data. Sertraline and desmethylsertraline showed high affinity for P-gp. The V(max)/K(m) values of sertraline (1.6 min(-1) x 10(-3)) and desmethylsertraline (1.4 min(-1) x 10(-3)) were comparable with that of verapamil (1.7 min(-1) x 10(-3)). Bupropion and its three metabolites showed very weak affinity for P-gp, with V(max)/K(m) values lower than 0.01 min(-1) x 10(-3). The results of the present study indicate that sertraline and desmethylsertraline have high affinity for P-gp, whereas bupropion and its three major metabolites TB, EB, and HB have very weak affinity for P-gp. These findings may help to explain observed drug-drug interactions among antidepressants.

  17. Effect of cocaine and sucrose withdrawal period on extinction behavior, cue-induced reinstatement, and protein levels of the dopamine transporter and tyrosine hydroxylase in limbic and cortical areas in rats

    PubMed Central

    Grimm, J.W.; Shaham, Y.; Hope, B.T.

    2010-01-01

    Lever pressing during tests for resistance to extinction and cue-induced reinstatement of cocaine seeking in rats progressively increases over the first 2 months of withdrawal. In the present report, we investigated the generality of these findings in rats trained to self-administer sucrose, a non-drug reinforcer. We also examined whether the time-dependent changes in cocaine seeking correlate with the levels of the dopamine transporter (DAT) and tyrosine hydroxylase (TH) proteins in the amygdala, nucleus accumbens, prefrontal cortex and orbitofrontal cortex. Rats were trained to self-administer cocaine (0.5 mg/kg/i.v. infusion) or 10% sucrose (0.2 ml/infusion into a liquid drop receptacle) for 10 days (6 h/day); each reward delivery was paired with a tone+light cue. Tests for cocaine seeking were conducted following 1 or 15 reward-free days. On the test day, rats were initially tested for resistance to extinction during 6–7 60-min extinction sessions in the absence of the tone–light cue, until they reached the extinction criterion of less than 15 responses/60 min. Subsequently, rats were tested for cue-induced reinstatement during a 60-min session in which each lever press led to a contingent presentation of the tone–light cue. Lever pressing during the tests for reward seeking was significantly greater on day 15 than on day 1 following withdrawal from both cocaine and sucrose self-administration training. The levels of DAT, but not TH, were greater in the prefrontal cortex of cocaine-trained rats than in sucrose-trained rats on both days 1 and 15 of withdrawal. The levels of DAT and TH in other brain areas were not altered following withdrawal from cocaine or sucrose self-administration. These data suggest that the withdrawal period can modulate reward seeking of both drug and non-drug reinforcers, and that alterations in DAT and TH levels in the brain regions examined do not mediate this effect. PMID:12394414

  18. Sucrose secreted by the engineered cyanobacterium and its fermentability

    NASA Astrophysics Data System (ADS)

    Duan, Yangkai; Luo, Quan; Liang, Feiyan; Lu, Xuefeng

    2016-10-01

    The unicellular cyanobacterium, Synechococcus elongatus PCC 7942 (Syn7942), synthesizes sucrose as the only compatible solute under salt stress. A series of engineered Syn7942 strains for sucrose production were constructed. The overexpression of the native sps (encoding a natively fused protein of sucrose phosphate synthase SPS and sucrose phosphate phosphatase SPP) in Syn7942 wild type caused a 93% improvement of sucrose productivity. The strain FL130 co-overexpressing sps and cscB (encoding a sucrose transporter) exhibited a 74% higher extracellular sucrose production than that overexpressing cscB only. Both results showed the significant improvement of sucrose productivity by the double functional protein SPS-SPP. Afterwards, FL130 was cultivated under a modified condition, and the cell-free culture medium containing 1.5 g L-1 sucrose was pre-treated with an acid hydrolysis technique. Cultivated with the neutralized hydrolysates as the starting media, two widely used microorganisms, Escherichia coli and Saccharomyces cerevisiae, showed a comparable growth with that in the control media supplemented with glucose. These results clearly demonstrated that the cell-free culture of sucrose-secreting cyanobacteria can be applied as starting media in microbial cultivation.

  19. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  20. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  1. Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

    PubMed Central

    Hara, T; Miyajima, A

    1992-01-01

    The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs. Images PMID:1582416

  2. ZrFsy1, a High-Affinity Fructose/H+ Symporter from Fructophilic Yeast Zygosaccharomyces rouxii

    PubMed Central

    Leandro, Maria José; Sychrová, Hana; Prista, Catarina; Loureiro-Dias, Maria C.

    2013-01-01

    Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H+ symporter, with Km 0.45±0.07 mM and Vmax 0.57±0.02 mmol h−1 (gdw) −1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system. PMID:23844167

  3. Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

    PubMed Central

    Echenique, J R; Arienti, H; Tolmasky, M E; Read, R R; Staneloni, R J; Crosa, J H; Actis, L A

    1992-01-01

    Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes. Images PMID:1447137

  4. The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

    SciTech Connect

    Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng; Garavito, R. Michael

    2014-10-02

    Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.

  5. Dual regulation of the Arabidopsis high-affinity root iron uptake system by local and long-distance signals.

    PubMed

    Vert, Grégory A; Briat, Jean-François; Curie, Catherine

    2003-06-01

    Regulation of the root high-affinity iron uptake system by whole-plant signals was investigated at the molecular level in Arabidopsis, through monitoring FRO2 and IRT1 gene expression. These two genes encode the root ferric-chelate reductase and the high-affinity iron transporter, respectively, involved in the iron deficiency-induced uptake system. Recovery from iron-deficient conditions and modulation of apoplastic iron pools indicate that iron itself plays a major role in the regulation of root iron deficiency responses at the mRNA and protein levels. Split-root experiments show that the expression of IRT1 and FRO2 is controlled both by a local induction from the root iron pool and through a systemic pathway involving a shoot-borne signal, both signals being integrated to tightly control production of the root iron uptake proteins. We also show that IRT1 and FRO2 are expressed during the day and down-regulated at night and that this additional control is overruled by iron starvation, indicating that the nutritional status prevails on the diurnal regulation. Our work suggests, for the first time to our knowledge, that like in grasses, the root iron acquisition in strategy I plants may also be under diurnal regulation. On the basis of the new molecular insights provided in this study and given the strict coregulation of IRT1 and FRO2 observed, we present a model of local and long-distance regulation of the root iron uptake system in Arabidopsis.

  6. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

    PubMed

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Rivero, Rosa M; Martínez, Vicente; Pardo, Jose M; Quintero, Francisco J; Rubio, Francisco

    2015-12-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

  7. Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high-affinity range of concentrations.

    PubMed

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

    2008-12-01

    The relative contribution of the high-affinity K(+) transporter AtHAK5 and the inward rectifier K(+) channel AtAKT1 to K(+) uptake in the high-affinity range of concentrations was studied in Arabidopsis thaliana ecotype Columbia (Col-0). The results obtained with wild-type lines, with T-DNA insertion in both genes and specific uptake inhibitors, show that AtHAK5 and AtAKT1 mediate the NH4+-sensitive and the Ba(2+)-sensitive components of uptake, respectively, and that they are the two major contributors to uptake in the high-affinity range of Rb(+) concentrations. Using Rb(+) as a K(+) analogue, it was shown that AtHAK5 mediates absorption at lower Rb(+) concentrations than AtAKT1 and depletes external Rb(+) to values around 1 muM. Factors such as the presence of K(+) or NH4+ during plant growth determine the relative contribution of each system. The presence of NH4+ in the growth solution inhibits the induction of AtHAK5 by K(+) starvation. In K(+)-starved plants grown without NH4+, both systems are operative, but when NH4+ is present in the growth solution, AtAKT1 is probably the only system mediating Rb(+) absorption, and the capacity of the roots to deplete Rb(+) is reduced.

  8. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    PubMed

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses.

  9. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity.

    PubMed

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K; Corey, David P

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.

  10. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

    PubMed Central

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906

  11. Selective high-affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2013-09-17

    This invention provides polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each binds different regions on the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  12. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  13. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    PubMed Central

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. Catalase<-->Abeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro. PMID:10567208

  14. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  15. Positron-labeled dopamine agonists for probing the high affinity states of dopamine subtype 2 receptors.

    PubMed

    Hwang, Dah-Ren; Narendran, Raj; Laruelle, Marc

    2005-01-01

    It is well documented that guanidine nucleotide-coupled dopamine subtype 2 receptors (D2) are configured in high and low affinity states for the dopamine agonist in vitro. However, it is still unclear whether these functional states exist in vivo. We hypothesized that positron-labeled D2 agonist and Positron Emission Tomography can be used to probe these functional states noninvasively. Recently, we demonstrated in nonhuman primates that N-[11C]propyl-norapomorphine (NPA), a full D2 agonist, is a suitable tracer for imaging the high affinity states of D2 receptors in vivo. We also developed kinetic modeling method to derive receptor parameters, such as binding potential (BP) and specific uptake ratios (V3''). When coupled with a dopamine releasing drug, amphetamine, NPA was found to be more sensitive than antagonist tracers, such as [11C]raclopride (RAC), to endogenous dopamine concentration changes (by about 42%). This finding suggests that NPA is a superior tracer for reporting endogenous DA concentration. In addition, the difference of the BP or V3'' of NPA and RAC under control and amphetamine challenge conditions could be used to estimate the functional states of D2 receptors in vivo. On the basis of our findings and the assumptions that NPA binds only to the high affinity states and RAC binds equally to both affinity states, we proposed that about 70% of the D2 receptors are configured in the high affinity states in vivo.

  16. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  17. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

    PubMed

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-03-03

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  18. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  19. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  20. Sucrose Synthase: Expanding Protein Function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  1. Sugar (sucrose) holograms

    NASA Astrophysics Data System (ADS)

    Ponce-Lee, E. L.; Olivares-Pérez, A.; Fuentes-Tapia, I.

    2004-06-01

    Computer holograms made with sugar crystals are reported. This material is well known as a good sweetener; the sugar from sugar cane or sugar beet (sucrose). These sweetener can be applied as honey "water and diluted sugar" easily on any substrate such as plastics or glasses without critical conditions for developed process. This step corresponds only to the cured sucrose as a photopolymer process. The maximum absorption spectra is localized at UV region λ=240 nm. We record with lithographic techniques some gratings, showing a good diffraction efficiency around 45%. This material has good resolution to make diffraction gratings. These properties are attractive because they open the possibility to make phase holograms on candies. Mainly the phase modulation is by refraction index.

  2. Oligosaccharides Derived from Sucrose

    NASA Astrophysics Data System (ADS)

    Monsan, Pierre F.; Ouarné, Francois

    Sucrose is a non-reducing disaccharide, consisting of an α-D-glucopyranosyl residue and a β-D-fructofuranosyl residue linked covalently by their respective anomeric carbons (α-D-glucopyranosyl-1,2-β-D-fructofuranoside). It is not just a simple disaccharide, among others: in fact, the energy of its glycosidic bond is higher than that of a usual glycosidic bond. It is equal to 27.6 kJ/mol, which is similar to the energy of a nucleotide-sugar bond as in UDP-glucose or ADP-glucose. This means that sucrose is a protected and activated form of D-glucose (as well as of D-fructose), which plays a key role in the metabolism of plants, for a wide variety of synthesis reactions.

  3. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  4. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  5. Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1985-01-01

    Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made. PMID:3977850

  6. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.

    PubMed

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C; Van Bergen En Henegouwen, Paul M P; Fernández, Luis Ángel

    2016-10-01

    Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.

  7. High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers

    SciTech Connect

    Campbell, K.P.; Sharp, A.; Strom, M.; Kahl, S.D.

    1986-05-01

    Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind (/sup 3/H)nitrendipine, (/sup 3/H)-nimodipine, (/sup 3/H)nisoldipine, and (/sup 3/H)PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while (/sup 3/H)verapamil, (/sup 3/H)diltiazem, and (/sup 3/H)trifluoperazine were not recognized. The average dissociation constant of the (/sup 3/H)nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of (/sup 3/H)nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify (/sup 3/H)nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace (/sup 3/H)nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

  8. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  9. Evidence for the presence of a sucrose carrier in immature sugar beet tap roots. [Beta vulgaris L

    SciTech Connect

    Lemoine, R.; Daie, J.; Wyse, R. )

    1988-02-01

    The objectives of this work were to determine the path of phloem unloading and if a sucrose carrier was present in young sugar beet (Beta vulgaris L.) taproots. The approach was to exploit the characteristics of the sucrose analog, 1{prime}-fluorosucrose (F-sucrose) which is a poor substrate for acid invertase but is a substrate for sucrose synthase. Ten millimolar each of ({sup 3}H) sucrose and ({sup 14}C)F-sucrose were applied in a 1:1 ratio to an abraded region of an attached leaf for 6 hours. ({sup 14}C)F-sucrose was translocated and accumulated in the roots at a higher rate than ({sup 3}H)sucrose. This was due to ({sup 3}H)sucrose hydrolysis along the translocation path. Presence of ({sup 3}H)hexose and ({sup 14}C)F-sucrose in the root apoplast suggested apoplastic sucrose unloading with its subsequent hydrolysis. Labeled F-sucrose uptake by root tissue discs exhibited biphasic kinetics and was inhibited by unlabeled sucrose, indicating that immature roots have the ability for carrier-mediated sucrose transport from the apoplast. Collectively, in vivo and in vitro data indicate that despite sucrose hydrolysis by the wall-bound invertase, sucrose hydrolysis is not entirely essential for sugar accumulation in this tissue.

  10. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    PubMed

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  11. Valproate is neuroprotective against malonate toxicity in rat striatum: an association with augmentation of high-affinity glutamate uptake.

    PubMed

    Morland, Cecilie; Boldingh, Karen Astrid; Iversen, Evy Grini; Hassel, Bjørnar

    2004-11-01

    The antiepileptic drug valproate (VPA) may be neuroprotective. We treated rats with VPA for 14 days (300 mg/kg twice daily) before intrastriatal injection of 1.5 micromol (1 M) of the succinate dehydrogenase inhibitor malonate. VPA-treated animals developed smaller lesions than control animals: 10 +/- 2 mm(3) versus 26 +/- 8 mm(3) (means +/- SD; P = 10(-4). Injection of NaCl that was equiosmolar with 1 M malonate caused lesions of only 1.2 +/- 0.4 mm(3) in control animals, whereas physiologic saline produced no lesion. VPA pretreatment reduced the malonate-induced extracellular accumulation of glutamate. This effect paralleled an increase in the striatal level of the glutamate transporter GLT, which augmented high-affinity glutamate uptake by 25%, as determined from the uptake of [(3)H] glutamate into striatal proteoliposomes. Malonate caused a 76% reduction in striatal adenosine triphosphate (ATP) content, but the glial, ATP-dependent formation of glutamine from radiolabeled glucose or glutamate was intact, indicating that glial ATP production supported uptake of glutamate. Striatal levels of HSP-70 and fos were reduced, and the levels of bcl-2 and phosphorylated extracellular signal-regulated kinase remained unaffected, but histone acetylation was increased by VPA treatment. The results suggest that augmentation of glutamate uptake may contribute importantly to VPA-mediated neuroprotection in striatum.

  12. Extraction of sucrose from molasses

    SciTech Connect

    Landis, A.M.

    1982-01-26

    Sucrose is extracted from molasses by passing an aqueous molasses solution over an adsorbent, e.g., calcined Al/sub 2/O/sub 3/-supported pyrolyzed C/sub 6/H/sub 6/. Thus, 10 mL molasses (approximately 46% solids) was run through a column containing 70 cubic centimetres above adsorbent with sucrose retention volume 21.4 and selectivity for sucrose - betaine 23.8.

  13. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  14. Selecting highly affine and well-expressed TCRs for gene therapy of melanoma.

    PubMed

    Jorritsma, Annelies; Gomez-Eerland, Raquel; Dokter, Maarten; van de Kasteele, Willeke; Zoet, Yvonne M; Doxiadis, Ilias I N; Rufer, Nathalie; Romero, Pedro; Morgan, Richard A; Schumacher, Ton N M; Haanen, John B A G

    2007-11-15

    A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

  15. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding.

    PubMed

    Rosilo, Henna; McKee, Jason R; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P; Ikkala, Olli; Kostiainen, Mauri A

    2014-10-21

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.

  16. The AVR4 elicitor protein of Cladosporium fulvum binds to fungal components with high affinity.

    PubMed

    Westerink, Nienke; Roth, Ronelle; Van den Burg, Harrold A; De Wit, Pierre J G M; Joosten, Matthieu H A J

    2002-12-01

    The interaction between tomato and the fungal pathogen Cladosporium fulvum complies with the gene-for-gene system. Strains of C. fulvum that produce race-specific elicitor AVR4 induce a hypersensitive response, leading to resistance, in tomato plants that carry the Cf-4 resistance gene. The mechanism of AVR4 perception was examined by performing binding studies with 125I-AVR4 on microsomal membranes of tomato plants. We identified an AVR4 high-affinity binding site (KD = 0.05 nM) which exhibited all the characteristics expected for ligand-receptor interactions, such as saturability, reversibility, and specificity. Surprisingly, the AVR4 high-affinity binding site appeared to originate from fungi present on infected tomato plants rather than from the tomato plants themselves. Detailed analysis showed that this fungus-derived, AVR4-specific binding site is heat- and proteinase K-resistant. Affinity crosslinking demonstrated that AVR4 specifically binds to a component of approximately 75 kDa that is of fungal origin. Our data suggest that binding of AVR4 to a fungal component or components is related to the intrinsic virulence function of AVR4 for C. fulvum.

  17. Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration.

    PubMed

    Kiosses, W B; Shattil, S J; Pampori, N; Schwartz, M A

    2001-03-01

    Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.

  18. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    SciTech Connect

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  19. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  20. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    PubMed Central

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  1. High-affinity interactions of ligands at recombinant Guinea pig 5HT7 receptors

    NASA Astrophysics Data System (ADS)

    Wilcox, R. E.; Ragan, J. E.; Pearlman, R. S.; Brusniak, M. Y.-. K.; Eglen, R. M.; Bonhaus, D. W.; Tenner, T. E., Jr.; Miller, J. D.

    2001-10-01

    The serotonin 5HT7 receptor has been implicated in numerous physiological and pathological processes from circadian rhythms [1] to depression and schizophrenia. Clonal cell lines heterologously expressing recombinant receptors offer good models for understanding drug-receptor interactions and development of quantitative structure-activity relationships (QSAR). Comparative Molecular Field Analysis (CoMFA) is an important modern QSAR procedure that relates the steric and electrostatic fields of a set of aligned compounds to affinity. Here, we utilized CoMFA to predict affinity for a number of high-affinity ligands at the recombinant guinea pig 5HT7 receptor. Using R-lisuride as the template, a final CoMFA model was derived using procedures similar to those of our recent papers [2, 3, 4] The final cross-validated model accounted for >85% of the variance in the compound affinity data, while the final non-cross validated model accounted for >99% of the variance. Model evaluation was done using cross-validation methods with groups of 5 ligands. Twenty cross-validation runs yielded an average predictive r2(q2) of 0.779 ± 0.015 (range: 0.669-0.867). Furthermore, 3D-chemical database search queries derived from the model yielded hit lists of promising agents with high structural similarity to the template. Together, these results suggest a possible basis for high-affinity drug action at 5HT7 receptors.

  2. A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding.

    PubMed

    Mercado, R; Hernández, J

    1992-09-18

    Axonal growth cone particles (AGCP) isolated from prenatal and postnatal rat brain had different high-affinity 5-HT uptake characteristics. In postnatal AGCP the uptake behaves as in the adult rat brain, while in the prenatal AGCP the uptake characteristics seem to be in a transitional stage. Also in prenatal AGCP we observed specific, high-affinity 5-HT binding sites. These results support the idea of an important role for 5-HT during axogenesis.

  3. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

    SciTech Connect

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. )

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  4. Location of high-affinity metal binding sites in the profile structure of the Ca+2-ATPase in the sarcoplasmic reticulum by resonance x-ray diffraction.

    PubMed Central

    Asturias, F J; Blasie, J K

    1991-01-01

    Resonance x-ray diffraction measurements on the lamellar diffraction from oriented multilayers of isolated sarcoplasmic reticulum (SR) membranes containing a small concentration of lanthanide (III) ions (lanthanide/protein molar ratio approximately 4) have allowed us to calculate both the electron density profile of the SR membrane and the separate electron density profile of the resonant lanthanide atoms bound to the membrane to a relatively low spatial resolution of approximately 40 A. Analysis of the membrane electron density profile and modeling of the separate low resolution lanthanide atom profile, using step-function electron density models based on the assumption that metal binding sites in the membrane profile are discrete and localized, resulted in the identification of a minimum of three such binding sites in the membrane profile. Two of these sites are low-affinity, low-occupancy sites identified with the two phospholipid polar headgroup regions of the lipid bilayer within the membrane profile. Up to 20% of the total lanthanide (III) ions bind to these low-affinity sites. The third site has relatively high affinity for lanthanide ion binding; its Ka is roughly an order of magnitude larger than that for the lower affinity polar headgroup sites. Approximately 80% of the total lanthanide ions present in the sample are bound to this high-affinity site, which is located in the "stalk" portion of the "headpiece" within the profile structure of the Ca+2 ATPase protein, approximately 12 A outside of the phospholipid polar headgroups on the extravesicular side of the membrane profile. Based on the nature of our results and on previous reports in the literature concerning the ability of lanthanide (III) ions to function as Ca+2 analogues for the Ca+2 ATPase we suggest that we have located a high-affinity metal binding site in the membrane profile which is involved in the active transport of Ca+2 ions across the SR membrane by the Ca+2 ATPase. PMID:1826221

  5. Translocation of labelled sucrose: A student exercise

    SciTech Connect

    Reiss, C. )

    1990-05-01

    Photosynthetic carbohydrates from the leaves are exported through the phloem to growing tips, roots, flowers and fruits. If sucrose labelled with {sup 14}C is applied to the leaves of bean plants, the pathway for sugar movement may be readily observed by autoradiography. Students apply the labelled sucrose during class time and return the next day to press their plants. During the next class, the pressed and dried plants are placed against X-ray film and left in the dark for four weeks. the film is then developed, examined for presence of label and compared to the pressed plants. Source to sink movement is clearly illustrated and information about the mechanism of phloem transport and loading is gained through experimental treatments, which include blocking the phloem pathway and inhibiting energy production.

  6. A linker peptide with high affinity towards silica-containing materials.

    PubMed

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

  7. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-07

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

  8. Protein unfolding as a switch from self-recognition to high-affinity client binding

    PubMed Central

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

  9. Glycan-based high-affinity ligands for toxins and pathogen receptors.

    PubMed

    Kulkarni, Ashish A; Weiss, Alison A; Iyer, Suri S

    2010-03-01

    Glycans decorate over 95% of the mammalian cell surface in the form of glycolipids and glycoproteins. Several toxins and pathogens bind to these glycans to enter the cells. Understanding the fundamentals of the complex interplay between microbial pathogens and their glycan receptors at the molecular level could lead to the development of novel therapeutics and diagnostics. Using Shiga toxin and influenza virus as examples, we describe the complex biological interface between host glycans and these infectious agents, and recent strategies to develop glycan-based high-affinity ligands. These molecules are expected to ultimately be incorporated into diagnostics and therapeutics, and can be used as probes to study important biological processes. Additionally, by focusing on the specific glycans that microbial pathogens target, we can begin to decipher the "glycocode" and how these glycans participate in normal and aberrant cellular communication.

  10. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

    PubMed Central

    Altschuler, Sarah E.; Lewis, Karen A.; Wuttke, Deborah S.

    2014-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization. PMID:25197549

  11. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

    PubMed Central

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-01-01

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

  12. Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex.

    PubMed

    Acuña Castroviejo, D; del Aguila, C M; Fernández, B; Gomar, M D; Castillo, J L

    1991-06-24

    The effect of the pineal gland on the ouabain high-affinity binding sites (Kd = 3.1 +/- 0.4 nM, Bmax = 246.4 +/- 18.4 fmol/mg protein) in rat cerebral cortex was studied. Pinealectomy increased Bmax (940.7 +/- 42.8 fmol/mg protein) and Kd (7.6 +/- 1.5 nM) while melatonin injection (100 micrograms/kg b.wt.) counteracted these effects, restoring kinetic parameters (Kd = 1.9 +/- 0.05 nM; Bmax = 262.2 +/- 29.6 fmol/mg prot) to control values. Melatonin activity on ouabain binding in vitro did not depend upon a direct effect on the binding sites themselves. However, in competition experiments, melatonin increased binding affinity of ouabain as shown by the decreased IC50 values.

  13. A complex water network contributes to high-affinity binding in an antibody-antigen interface.

    PubMed

    Marino, S F; Olal, D; Daumke, O

    2016-03-01

    This data article presents an analysis of structural water molecules in the high affinity interaction between a potent tumor growth inhibiting antibody (fragment), J22.9-xi, and the tumor marker antigen CD269 (B cell maturation antigen, BCMA). The 1.89 Å X-ray crystal structure shows exquisite details of the binding interface between the two molecules, which comprises relatively few, mostly hydrophobic, direct contacts but many indirect interactions over solvent waters. These are partly or wholly buried in, and therefore part of, the interface. A partial description of the structure is included in an article on the tumor inhibiting effects of the antibody: "Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma", Mol. Oncol. 9 (7) (2015) pp. 1348-58.

  14. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

    NASA Astrophysics Data System (ADS)

    Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

    2014-09-01

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface

  15. Experimental conditions can obscure the second high-affinity site in LeuT.

    PubMed

    Quick, Matthias; Shi, Lei; Zehnpfennig, Britta; Weinstein, Harel; Javitch, Jonathan A

    2012-01-15

    Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

  16. Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes.

    PubMed

    Rosin, Carina; Ordieres, María Graciela López; Arnaiz, Georgina Rodríguez de Lores

    2011-12-10

    Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

  17. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    SciTech Connect

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  18. The integration of genomic and structural information in the development of high affinity plasmepsin inhibitors.

    PubMed

    Nezami, Azin; Freire, Ernesto

    2002-12-04

    The plasmepsins are key enzymes in the life cycle of the Plasmodium parasites responsible for malaria. Since plasmepsin inhibition leads to parasite death, these enzymes have been acknowledged to be important targets for the development of new antimalarial drugs. The development of effective plasmepsin inhibitors, however, is compounded by their genomic diversity which gives rise not to a unique target for drug development but to a family of closely related targets. Successful drugs will have to inhibit not one but several related enzymes with high affinity. Structure-based drug design against heterogeneous targets requires a departure from the classic 'lock-and-key' paradigm that leads to the development of conformationally constrained molecules aimed at a single target. Drug molecules designed along those principles are usually rigid and unable to adapt to target variations arising from naturally occurring genetic polymorphisms or drug-induced resistant mutations. Heterogeneous targets need adaptive drug molecules, characterised by the presence of flexible elements at specific locations that sustain a viable binding affinity against existing or expected polymorphisms. Adaptive ligands have characteristic thermodynamic signatures that distinguish them from their rigid counterparts. This realisation has led to the development of rigorous thermodynamic design guidelines that take advantage of correlations between the structure of lead compounds and the enthalpic and entropic components of the binding affinity. In this paper, we discuss the application of the thermodynamic approach to the development of high affinity (K(i) - pM) plasmepsin inhibitors. In particular, a family of allophenylnorstatine-based compounds is evaluated for their potential to inhibit a wide spectrum of plasmepsins.

  19. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    SciTech Connect

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  20. A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata.

    PubMed

    Srivastava, Vivek Kumar; Suneetha, Korivi Jyothiraj; Kaur, Rupinder

    2014-10-01

    Iron is an essential nutrient for all living organisms and human pathogens employ a battery of factors to scavenge iron from the high-affinity iron-binding host proteins. In the present study, we have elucidated, via a candidate gene approach, major iron acquisition and homoeostatic mechanisms operational in an opportunistic human fungal pathogen Candida glabrata. Phenotypic, biochemical and molecular analysis of a set of 13 C. glabrata strains, deleted for proteins potentially implicated in iron metabolism, revealed that the high-affinity reductive iron uptake system is required for utilization of alternate carbon sources and for growth under both in vitro iron-limiting and in vivo conditions. Furthermore, we show for the first time that the cysteine-rich CFEM (common in fungal extracellular membranes) domain-containing cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3, are essential for maintenance of intracellular iron content, adherence to epithelial cells and virulence. Consistent with their roles in iron homoeostasis, mitochondrial aconitase activity was lower and higher in mutants disrupted for high-affinity iron transport, and haemolysin respectively. Additionally, we present evidence that the mitochondrial frataxin, CgYfh1, is pivotal to iron metabolism. Besides yielding insights into major in vitro and in vivo iron acquisition strategies, our findings establish high-affinity iron uptake mechanisms as critical virulence determinants in C. glabrata.

  1. The Ketamine Analogue Methoxetamine and 3- and 4-Methoxy Analogues of Phencyclidine Are High Affinity and Selective Ligands for the Glutamate NMDA Receptor

    PubMed Central

    Roth, Bryan L.; Gibbons, Simon; Arunotayanun, Warunya; Huang, Xi-Ping; Setola, Vincent; Treble, Ric; Iversen, Les

    2013-01-01

    In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as ‘designer drugs’ and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects. PMID:23527166

  2. Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield.

    PubMed

    Basso, Thiago O; de Kok, Stefan; Dario, Marcelo; do Espirito-Santo, Júlio Cézar A; Müller, Gabriela; Schlölg, Paulo S; Silva, Carlos P; Tonso, Aldo; Daran, Jean-Marc; Gombert, Andreas K; van Maris, Antonius J A; Pronk, Jack T; Stambuk, Boris U

    2011-11-01

    Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

  3. Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production

    PubMed Central

    Dusotoit-Coucaud, Anaïs; Brunel, Nicole; Kongsawadworakul, Panida; Viboonjun, Unchera; Lacointe, André; Julien, Jean-Louis; Chrestin, Hervé; Sakr, Soulaïman

    2009-01-01

    Background and Aims The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel®) on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described. Methods Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel. Key Results Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application. Conclusion The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees. PMID:19567416

  4. Expression patterns of Brassica napus genes implicate IPT, CKX, sucrose transporter, cell wall invertase, and amino acid permease gene family members in leaf, flower, silique, and seed development.

    PubMed

    Song, Jiancheng; Jiang, Lijun; Jameson, Paula Elizabeth

    2015-08-01

    Forage brassica (Brassica napus cv. Greenland) is bred for vegetative growth and biomass production, while its seed yield remains to be improved for seed producers without affecting forage yield and quality. Cytokinins affect seed yield by influencing flower, silique and seed number, and seed size. To identify specific cytokinin gene family members as targets for breeding, as well as genes associated with yield and/or quality, a B. napus transcriptome was obtained from a mixed sample including leaves, flower buds and siliques of various stages. Gene families for cytokinin biosynthesis (BnIPT1, 2, 3, 5, 7, 8 and 9), cytokinin degradation (BnCKX1 to BnCKX7), cell wall invertase (BnCWINV1 to BnCWINV6), sugar transporter (BnSUT1 to BnSUT6) and amino acid permease (BnAAP1 to BnAAP8) were identified. As B. napus is tetraploid, homoeologues of each gene family member were sought. Using multiple alignments and phylogenetic analysis, the parental genomes of the two B. napus homoeologues could be differentiated. RT-qPCR was then used to determine the expression of gene family members and their homoeologues in leaves, flowers, siliques and seeds of different developmental stages. The expression analysis showed both temporal and organ-specific expression profiles among members of these multi-gene families. Several pairs of homoeologues showed differential expression, both in terms of level of expression and differences in temporal or organ-specificity. BnCKX2 and 4 were identified as targets for TILLING, EcoTILLING and MAS.

  5. Expression patterns of Brassica napus genes implicate IPT, CKX, sucrose transporter, cell wall invertase, and amino acid permease gene family members in leaf, flower, silique, and seed development

    PubMed Central

    Song, Jiancheng; Jiang, Lijun; Jameson, Paula Elizabeth

    2015-01-01

    Forage brassica (Brassica napus cv. Greenland) is bred for vegetative growth and biomass production, while its seed yield remains to be improved for seed producers without affecting forage yield and quality. Cytokinins affect seed yield by influencing flower, silique and seed number, and seed size. To identify specific cytokinin gene family members as targets for breeding, as well as genes associated with yield and/or quality, a B. napus transcriptome was obtained from a mixed sample including leaves, flower buds and siliques of various stages. Gene families for cytokinin biosynthesis (BnIPT1, 2, 3, 5, 7, 8 and 9), cytokinin degradation (BnCKX1 to BnCKX7), cell wall invertase (BnCWINV1 to BnCWINV6), sugar transporter (BnSUT1 to BnSUT6) and amino acid permease (BnAAP1 to BnAAP8) were identified. As B. napus is tetraploid, homoeologues of each gene family member were sought. Using multiple alignments and phylogenetic analysis, the parental genomes of the two B. napus homoeologues could be differentiated. RT-qPCR was then used to determine the expression of gene family members and their homoeologues in leaves, flowers, siliques and seeds of different developmental stages. The expression analysis showed both temporal and organ-specific expression profiles among members of these multi-gene families. Several pairs of homoeologues showed differential expression, both in terms of level of expression and differences in temporal or organ-specificity. BnCKX2 and 4 were identified as targets for TILLING, EcoTILLING and MAS. PMID:25873685

  6. Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors

    PubMed Central

    Ballesteros-Yáñez, Inmaculada; Benavides-Piccione, Ruth; Bourgeois, Jean-Pierre; Changeux, Jean-Pierre; DeFelipe, Javier

    2010-01-01

    The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex. PMID:20534523

  7. A 45-amino acid scaffold mined from the Protein Data Bank for high affinity ligand engineering

    PubMed Central

    Kruziki, Max A.; Bhatnagar, Sumit; Woldring, Daniel R.; Duong, Vandon T.; Hackel, Benjamin J.

    2015-01-01

    Summary Small protein ligands can provide superior physiological distribution versus antibodies and improved stability, production, and specific conjugation. Systematic evaluation of the Protein Data Bank identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α-helix opposite a β-sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 108 mutants and directed evolution towards four targets yielded target-specific binders with affinities as strong as 200 ±100 pM, Tm’s from 65 ±3 °C to 80 ±1 °C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ±8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold. PMID:26165154

  8. Role of constraint in catalysis and high-affinity binding by proteins.

    PubMed Central

    Vanselow, Donald G

    2002-01-01

    Using a model for catalysis of a dynamic equilibrium, the role of constraint in catalysis is quantified. The intrinsic rigidity of proteins is shown to be insufficient to constrain the activated complexes of enzymes, irrespective of the mechanism. However, when minimization of the surface excess free energy of water surrounding a protein is considered, model proteins can be designed with regions of sufficient rigidity. Structures can be designed to focus surface tension or hydrophobic attraction as compressive stress. A monomeric structure has a limited ability to concentrate compressive stress and constrain activated complexes. Oligomeric or multidomain proteins, with domains surrounding a rigid core, have unlimited ability to concentrate stress, provided there are at least four domains. Under some circumstances, four is the optimum number, which could explain the frequency of tetrameric enzymes in nature. The minimum compressive stress in oligomers increases with the square of the radius. For tetramers of similar size to natural enzymes, this stress agrees reasonably well with that needed to constrain the activated complex. A similar principle applies to high affinity binding proteins. The models explain the trigonal pyramidal shape of fibroblast growth factor and provide a basis for interpretation of protein crystal structures. PMID:11964220

  9. High-affinity DNA base analogs as supramolecular, nanoscale promoters of macroscopic adhesion.

    PubMed

    Anderson, Cyrus A; Jones, Amanda R; Briggs, Ellen M; Novitsky, Eric J; Kuykendall, Darrell W; Sottos, Nancy R; Zimmerman, Steven C

    2013-05-15

    Adhesion phenomena are essential to many biological processes and to synthetic adhesives and manufactured coatings and composites. Supramolecular interactions are often implicated in various adhesion mechanisms. Recently, supramolecular building blocks, such as synthetic DNA base-pair mimics, have drawn attention in the context of molecular recognition, self-assembly, and supramolecular polymers. These reversible, hydrogen-bonding interactions have been studied extensively for their adhesive capabilities at the nano- and microscale, however, much less is known about their utility for practical adhesion in macroscopic systems. Herein, we report the preparation and evaluation of supramolecular coupling agents based on high-affinity, high-fidelity quadruple hydrogen-bonding units (e.g., DAN·DeUG, Kassoc = 10(8) M(-1) in chloroform). Macroscopic adhesion between polystyrene films and glass surfaces modified with 2,7-diamidonaphthyridine (DAN) and ureido-7-deazaguanine (DeUG) units was evaluated by mechanical testing. Structure-property relationships indicate that the designed supramolecular interaction at the nanoscale plays a key role in the observed macroscopic adhesive response. Experiments probing reversible adhesion or self-healing properties of bulk samples indicate that significant recovery of initial strength can be realized after failure but that the designed noncovalent interaction does not lead to healing during the process of adhesion loss.

  10. Enhanced Membrane Pore Formation through High-Affinity Targeted Antimicrobial Peptides

    PubMed Central

    Arnusch, Christopher J.; Pieters, Roland J.; Breukink, Eefjan

    2012-01-01

    Many cationic antimicrobial peptides (AMPs) target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted. PMID:22768121

  11. Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.

    PubMed

    Konitsiotis, Antonios D; Raynal, Nicolas; Bihan, Dominique; Hohenester, Erhard; Farndale, Richard W; Leitinger, Birgit

    2008-03-14

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.

  12. Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

    PubMed Central

    Gherghe, Cristina; Lombo, Tania; Leonard, Christopher W.; Datta, Siddhartha A. K.; Bess, Julian W.; Gorelick, Robert J.; Rein, Alan; Weeks, Kevin M.

    2010-01-01

    All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs—only four nucleotides per genomic RNA—reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs. PMID:20974908

  13. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  14. Evolved Streptavidin Mutants Reveal Key Role of Loop Residue in High-affinity Binding

    SciTech Connect

    M Magalhaes; C Melo Czekster; R Guan; V Malashkevich; S Almo; M Levy

    2011-12-31

    We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a {approx}10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.

  15. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

    PubMed Central

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

  16. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin.

    PubMed

    Shulman, Ziv; Shinder, Vera; Klein, Eugenia; Grabovsky, Valentin; Yeger, Orna; Geron, Erez; Montresor, Alessio; Bolomini-Vittori, Matteo; Feigelson, Sara W; Kirchhausen, Tomas; Laudanna, Carlo; Shakhar, Guy; Alon, Ronen

    2009-03-20

    Endothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.

  17. Characterization of a high affinity cocaine binding site in rat brain

    SciTech Connect

    Calligaro, D.; Eldefrawi, M.

    1986-03-05

    Binding of (/sup 3/H)cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of (/sup 3/H)cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95/sup 0/C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of (/sup 3/H)cocaine (15 nM) was inhibited by increasing concentrations of Na/sup +/ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific (/sup 3/H)cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC/sub 50/ = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting (/sup 3/H)cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC/sub 50/ values below ..mu..M concentrations.

  18. Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity

    PubMed Central

    Kang, Dezhi; Wang, Jiangjie; Zhang, Weiyun; Song, Yanling; Li, Xilan; Zou, Yuan; Zhu, Mingtao; Zhu, Zhi; Chen, Fuyong; Yang, Chaoyong James

    2012-01-01

    Background Glioblastoma is the most common and most lethal form of brain tumor in human. Unfortunately, there is still no effective therapy to this fatal disease and the median survival is generally less than one year from the time of diagnosis. Discovery of ligands that can bind specifically to this type of tumor cells will be of great significance to develop early molecular imaging, targeted delivery and guided surgery methods to battle this type of brain tumor. Methodology/Principal Findings We discovered two target-specific aptamers named GBM128 and GBM131 against cultured human glioblastoma cell line U118-MG after 30 rounds selection by a method called cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). These two aptamers have high affinity and specificity against target glioblastoma cells. They neither recognize normal astraglial cells, nor do they recognize other normal and cancer cell lines tested. Clinical tissues were also tested and the results showed that these two aptamers can bind to different clinical glioma tissues but not normal brain tissues. More importantly, binding affinity and selectivity of these two aptamers were retained in complicated biological environment. Conclusion/Significance The selected aptamers could be used to identify specific glioblastoma biomarkers. Methods of molecular imaging, targeted drug delivery, ligand guided surgery can be further developed based on these ligands for early detection, targeted therapy, and guided surgery of glioblastoma leading to effective treatment of glioblastoma. PMID:23056171

  19. Expression of high affinity folate receptor in breast cancer brain metastasis.

    PubMed

    Leone, José Pablo; Bhargava, Rohit; Theisen, Brian K; Hamilton, Ronald L; Lee, Adrian V; Brufsky, Adam M

    2015-10-06

    High affinity folate receptor (HFR) can be overexpressed in breast cancer and is associated with poor prognosis, however the expression in breast cancer brain metastases (BCBM) is unknown. The aim of this study was to analyze the rate of HFR expression in BCBM and its role in the prognosis of this high-risk cohort. We analyzed 19 brain metastasis (BM) and 13 primary tumors (PT) from a total of 25 patients. HFR status was assessed by immunohistochemistry. Median follow-up was 4.2 years (range 0.6-18.5). HFR was positive in 4/19 BM (21.1%) and in 1/13 PT (7.7%). Positive samples had low H-scores (range 1-50). 56% of patients had apocrine differentiation. OS was similar between patients with positive HFR (median OS 48 months) and negative HFR (median OS 69 months) (P = 0.25); and between patients with apocrine differentiation (median OS 63 months) and those without apocrine differentiation (median OS 69 months) (P = 0.49). To the best of our knowledge, this is the first analysis of HFR expression in BCBM. While previous studies associated the presence of HFR with worse prognosis; in our cohort HFR was positive in only 21.1% of BM with low levels of positivity. Neither HFR nor apocrine features had impact in OS.

  20. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

    PubMed

    Bedzyk, W D; Weidner, K M; Denzin, L K; Johnson, L S; Hardman, K D; Pantoliano, M W; Asel, E D; Voss, E W

    1990-10-25

    Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

  1. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    SciTech Connect

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  2. Effects of lead on the kidney: Roles of high-affinity lead-binding proteins

    SciTech Connect

    Fowler, B.A. ); DuVal, G. )

    1991-02-01

    Lead-induced nephropathy produces both tubular and interstitial manifestations of cell injury, but the pathophysiology of these lesions is not completely understood. Delineation of the molecular factors underlying renal handling of lead is one of central importance in understanding the mechanisms of renal cell injury from this agent. Recent studies from this laboratory have identified several distinct high-affinity lead-binding proteins (PbBP) from rat kidney and brain that appear to play critical roles in the intracellular bioavailability of lead to several essential cellular processes in these target tissues at low dose levels. These studies have also shown that the real PbBP is selectively localized in only certain nephrons and only specific segments of the renal proximal tubule. The striking nephron and cell-type specificity of the localization reaction could result from physoiological differences in nephron functional activity or selective molecular uptake mechanisms/metabolism differences that act to define target cell populations in the kidney. In addition, other preliminary studies have shown that short-term, high-dose lead exposure produces increased excretion of this protein into the urine with concomitant decreases in renal concentrations.

  3. The molecular basis of talin2’s high affinity toward β1-integrin

    PubMed Central

    Yuan, Yaxia; Li, Liqing; Zhu, Yanyan; Qi, Lei; Azizi, Latifeh; Hytönen, Vesa P.; Zhan, Chang-Guo; Huang, Cai

    2017-01-01

    Talin interacts with β-integrin tails and actin to control integrin activation, thus regulating focal adhesion dynamics and cell migration. There are two talin genes, Tln1 and Tln2, which encode talin1 and talin2, and it is generally believed that talin2 functions redundantly with talin1. However, we show here that talin2 has a higher affinity to β1-integrin tails than talin1. Mutation of talin2 S339 to leucine, which can cause Fifth Finger Camptodactyly, a human genetic disease, completely disrupted its binding to β–integrin tails. Also, substitution of talin1 C336 with Ser enhanced the affinity of talin1, whereas substitution of talin2 S339 with Cys diminished that of talin2. Further computational modeling analysis shows that talin2 S339 formed a hydrogen bond with E353, which is critical for inducing key hydrogen bonds between talin2 N326 and β1-integrin R760, and between talin2 K327 and β1-integrin D759. Mutation at any of these residues significantly diminished the interaction of talin2 with β1- integrin tails. These hydrogen bonds were not observed in talin1/β1-integrin, but did exist in talin1C336S/β1-integrin complex. These results suggest that talin2 S339 forms a hydrogen bond with E353 to mediate its high affinity to β1-integrin. PMID:28155884

  4. Comparison of three high affinity SPECT radiotracers for the dopamine D2 receptor.

    PubMed

    al-Tikriti, M S; Baldwin, R M; Zea-Ponce, Y; Sybirska, E; Zoghbi, S S; Laruelle, M; Malison, R T; Kung, H F; Kessler, R M; Charney, D S

    1994-02-01

    The regional brain distribution and pharmacological specificity of three high affinity tracers for the dopamine (DA) D2 receptor: [123I]IBF, [123I]epidepride, and [123I]2'-ISP were assessed by SPECT imaging of non-human primates. The ratios of striatal-to-occipital activities at the time of peak striatal uptake were 2.2, 6.3 and 1.7, respectively. From the peak striatal activities, washout rates were 33, 4 and 16%/h for [123I]IBF, [123I]epidepride and [123I]2'-ISP, respectively. The reversibility of the striatal uptake of all three agents was demonstrated by the rapid displacement induced by the dopamine D2 selective antipsychotic agent raclopride, which increased washout rates to 96, 58 and 43%/h. The administration of d-amphetamine, which induces release of dopamine, had no noticeable effect on [123I]epidepride but increased the washout rate of [123I]IBF. These results suggest that, among these three agents, [123I]epidepride is the superior tracer for in vivo displacement studies because of its slow washout and high target-to-background ratios. However, for tracer kinetic modeling, [123I]IBF may be the superior agent because of its early time of peak uptake and its higher target-to-background ratios than [123I]2'-ISP.

  5. New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling.

    PubMed

    Bambouskova, Monika; Polakovicova, Iva; Halova, Ivana; Goel, Gautam; Draberova, Lubica; Bugajev, Viktor; Doan, Aivi; Utekal, Pavol; Gardet, Agnes; Xavier, Ramnik J; Draber, Petr

    2016-05-01

    Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

  6. High affinity truncated DNA aptamers for the development of fluorescence based progesterone biosensors.

    PubMed

    Alhadrami, Hani A; Chinnappan, Raja; Eissa, Shimaa; Rahamn, Anas Abdel; Zourob, Mohammed

    2017-02-24

    Aptamers have shown a number of potential applications in sensing and therapeutic due to the high affinity and specificity towards their target molecules. Not all the nucleotides in the full length aptamers are involved in the binding with their targets. The non-binding domain of the aptamer may affect the binding affinity of the aptamer-target complex. Mapping the aptamer binding region could increase the affinity and the specificity. In this paper, we designed aptamer-based fluorescence sensors from a truncated progesterone (P4) aptamer. Then, fluorescein and quencher labelled aptamer complementary oligonucleotide sequences were hybridized to the truncated aptamer at different sites to form duplex structures. We used fluorescence-quencher pair displacement assay upon progesterone binding for the determination of P4. One of the truncated sequences has shown high binding affinity with 16 fold increase in the dissociation constant, Kd (2.1 nM) compared to the original aptamer. The aptasensor was highly selective for P4 against similar compounds such as 17-β estradiol, bisphenol-A and vitamin D. The sensor has been applied for the detection of P4 in spiked tap water and in urine samples showing good recovery. This new developed aptamer-based fluorescence biosensor can be applied in food, pharmaceutical, and clinical industries.

  7. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    SciTech Connect

    Raza, H.; Chung, W.L.; Mukhtar, H. )

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  8. High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

    PubMed Central

    Vernhes, Emeline; Renouard, Madalena; Gilquin, Bernard; Cuniasse, Philippe; Durand, Dominique; England, Patrick; Hoos, Sylviane; Huet, Alexis; Conway, James F.; Glukhov, Anatoly; Ksenzenko, Vladimir; Jacquet, Eric; Nhiri, Naïma; Zinn-Justin, Sophie; Boulanger, Pascale

    2017-01-01

    Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments. PMID:28165000

  9. Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.

    PubMed

    Papadakos, Grigorios; Sharma, Amit; Lancaster, Lorna E; Bowen, Rebecca; Kaminska, Renata; Leech, Andrew P; Walker, Daniel; Redfield, Christina; Kleanthous, Colin

    2015-04-29

    The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.

  10. Peptide array-based characterization and design of ZnO-high affinity peptides.

    PubMed

    Okochi, Mina; Sugita, Tomoya; Furusawa, Seiji; Umetsu, Mitsuo; Adschiri, Tadafumi; Honda, Hiroyuki

    2010-08-15

    Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self-assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO-binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO-binding peptide, 125 spot-synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO-binding sites were found as 6-mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles.

  11. High affinity group III mGluRs regulate mossy fiber input to CA3 interneurons.

    PubMed

    Cosgrove, Kathleen E; Meriney, Stephen D; Barrionuevo, Germán

    2011-12-01

    Stratum lacunosum-moleculare interneurons (L-Mi) in hippocampal area CA3 target the apical dendrite of pyramidal cells providing feedforward inhibition. Here we report that selective activation of group III metabotropic glutamate receptors (mGluRs) 4/8 with L(+)-2-amino-4-phosphnobytyric acid (L-AP4; 10 μM) decreased the probability of glutamate release from the mossy fiber (MF) terminals synapsing onto L-Mi. Consistent with this interpretation, application of L-AP4 in the presence of 3 mM strontium decreased the frequency of asynchronous MF EPSCs in L-Mi. Furthermore, the dose response curve showed that L-AP4 at 400 μM produced no further decrease in MF EPSC amplitude compared with 20 μM L-AP4, indicating the lack of mGluRs 7 at these MF terminals. We also found that one mechanism of mGluRs 4/8-mediated inhibition of release is linked to N-type voltage gated calcium channels at MF terminals. Application of the group III mGluR antagonist MSOP (100 μM) demonstrated that mGluRs 4/8 are neither tonically active nor activated by low and moderate frequencies of activity. However, trains of stimuli to the MF at 20 and 40 Hz delivered during the application of MSOP revealed a relief of inhibition of transmitter release and an increase in the overall probability of action potential firing in the postsynaptic L-Mi. Interestingly, the time to first action potential was significantly shorter in the presence of MSOP, indicating that mGluR 4/8 activation delays L-Mi firing in response to MF activity. Taken together, our data demonstrate that the timing and probability of action potentials in L-Mi evoked by MF synaptic input is regulated by the activation of presynaptic high affinity group III mGluRs.

  12. Devices and approaches for generating specific high-affinity nucleic acid aptamers

    NASA Astrophysics Data System (ADS)

    Szeto, Kylan; Craighead, Harold G.

    2014-09-01

    High-affinity and highly specific antibody proteins have played a critical role in biological imaging, medical diagnostics, and therapeutics. Recently, a new class of molecules called aptamers has emerged as an alternative to antibodies. Aptamers are short nucleic acid molecules that can be generated and synthesized in vitro to bind to virtually any target in a wide range of environments. They are, in principal, less expensive and more reproducible than antibodies, and their versatility creates possibilities for new technologies. Aptamers are generated using libraries of nucleic acid molecules with random sequences that are subjected to affinity selections for binding to specific target molecules. This is commonly done through a process called Systematic Evolution of Ligands by EXponential enrichment, in which target-bound nucleic acids are isolated from the pool, amplified to high copy numbers, and then reselected against the desired target. This iterative process is continued until the highest affinity nucleic acid sequences dominate the enriched pool. Traditional selections require a dozen or more laborious cycles to isolate strongly binding aptamers, which can take months to complete and consume large quantities of reagents. However, new devices and insights from engineering and the physical sciences have contributed to a reduction in the time and effort needed to generate aptamers. As the demand for these new molecules increases, more efficient and sensitive selection technologies will be needed. These new technologies will need to use smaller samples, exploit a wider range of chemistries and techniques for manipulating binding, and integrate and automate the selection steps. Here, we review new methods and technologies that are being developed towards this goal, and we discuss their roles in accelerating the availability of novel aptamers.

  13. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    SciTech Connect

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

  14. Can we accurately quantify nanoparticle associated proteins when constructing high-affinity MRI molecular imaging probes?

    PubMed

    Rimkus, Gabriella; Bremer-Streck, Sibylle; Grüttner, Cordula; Kaiser, Werner Alois; Hilger, Ingrid

    2011-01-01

    Targeted magnetic resonance contrast agents (e.g. iron oxide nanoparticles) have the potential to become highly selective imaging tools. In this context, quantification of the coupled amount of protein is essential for the design of antibody- or antibody fragment-conjugated nanoparticles. Nevertheless, the presence of magnetic iron oxide nanoparticles is still an unsolved problem for this task. The aim of the present work was to clarify whether proteins can be reliably quantified directly in the presence of magnetic iron oxide nanoparticles without the use of fluorescence or radioactivity. Protein quantification via Bradford was not influenced by the presence of magnetic iron oxide nanoparticles (0-17.2 mmol Fe l(-1) ). Instead, bicinchoninic acid based assay was, indeed, distinctly affected by the presence of nanoparticle-iron in suspension (0.1-17.2 mmol Fe l(-1) ), although the influence was linear. This observation allowed for adequate mathematical corrections with known iron content of a given nanoparticle. The applicability of our approach was demonstrated by the determination of bovine serum albumin (BSA) content coupled to dextrane-coated magnetic nanoparticles, which was found with the QuantiPro Bicinchoninic acid assay to be of 1.5 ± 0.2 µg BSA per 1 mg nanoparticle. Both Bradford and bicinchoninic acid assay protein assays allow for direct quantification of proteins in the presence of iron oxide containing magnetic nanoparticles, without the need for the introduction of radioactivity or fluorescence modules. Thus in future it should be possible to make more precise estimations about the coupled protein amount in high-affinity targeted MRI probes for the identification of specific molecules in living organisms, an aspect which is lacking in corresponding works published so far. Additionally, the present protein coupling procedures can be drastically improved by our proposed protein quantification method.

  15. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  16. A functionally active presynaptic high-affinity kainate receptor in the rat hippocampal CA3 subregion.

    PubMed

    Malva, J O; Ambrósio, A F; Cunha, R A; Ribeiro, J A; Carvalho, A P; Carvalho, C M

    1995-02-09

    We studied the modulation of the intracellular free calcium concentrations ([Ca2+]i) by kainate/AMPA receptor activation in synaptosomes isolated from whole rat hippocampus, or from its CA1, CA3 or dentate gyrus subregions. The receptor was activated either by 100 microM S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionic acid (AMPA) (EC50 = 26.6 +/- 4.9 microM) or by 100 microM kainate (EC50 = 0.81 +/- 0.1 microM), but the effects of these agonists were not additive. The response to either AMPA or kainate was competitively inhibited by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dioxine. Higher [Ca2+]i responses to 100 microM AMPA or to 100 microM kainate were observed in the CA3 subregion (43.2 +/- 2.5 nM or 42.8 +/- 2.3 nM, respectively) than in the whole hippocampus (22.4 +/- 1.1 nM or 22.4 +/- 1.6, respectively), in the CA1 subregion (26.4 +/- 1.1 nM or 26.6 +/- 2.6 nM, respectively) or in dentate gyrus (24.6 +/- 1.4 nM or 21.5 +/- 1.0 nM, respectively). These results indicate that the CA3 subregion of the hippocampus is enriched in a presynaptic high-affinity kainate receptor which modulates the [Ca2+]i in nerve terminals.

  17. In vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers.

    PubMed

    Elshafey, Reda; Siaj, Mohamed; Zourob, Mohammed

    2014-09-16

    Contamination of freshwater with cyanotoxin cylindrospermopsin (CYN) represents a significant global concern for public health. The sensitive detection of CYN is necessary to effectively manage and control the treatment of water resources. Here we report a novel, highly sensitive label-free aptasensor for CYN analysis, using aptamers as specific receptors. We have selected the DNA aptamers from a diverse random library using the in vitro screening SELEX approach. The aptamers exhibited high affinity for CYN with Kd of nanomolar range. One aptamer exhibited conformational change upon CYN recognition (CD analysis) and was used to fabricate the label-free impedimetric aptasensor for CYN. A self-assembled monolayer from a disulfide-derivatized aptamer was formed on a gold electrode to fabricate the aptasensor. Upon CYN capturing to the aptasensor surface, a marked drop in the electron transfer resistance was obtained, which was used as the principle of detection of CYN. This resulted from the aptamer's conformational change induced by CYN recognition. The present aptasensor could detect CYN with the limit of detection as low as 100 pM and a wide linear range of 0.1 to 80 nM. When mounted on the gold surface, the aptamer exhibited a lower dissociation constant for CYN than that observed in the fluorescence assay, implying that the anchoring of the aptamer on the Au surface improved its affinity to CYN. Moreover, the aptasensor showed high specificity toward other coexistent cyanobacterial toxins of microcystin-LR and Anatoxin-a. Further biosensor designs will be generated using those aptamers for simple and sensitive CYN monitoring.

  18. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  19. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors.

    PubMed

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging.

  20. High-affinity σ1 protein agonist reduces clinical and pathological signs of experimental autoimmune encephalomyelitis

    PubMed Central

    Oxombre, B; Lee-Chang, C; Duhamel, A; Toussaint, M; Giroux, M; Donnier-Maréchal, M; Carato, P; Lefranc, D; Zéphir, H; Prin, L; Melnyk, P; Vermersch, P

    2015-01-01

    Background and Purpose Selective agonists of the sigma-1 receptor (σ1 protein) are generally reported to protect against neuronal damage and modulate oligodendrocyte differentiation. Human and rodent lymphocytes possess saturable, high-affinity binding sites for compounds binding to the σ1 protein and potential immunomodulatory properties have been described for σ1 protein ligands. Experimental autoimmune encephalomyelitis (EAE) is recognized as a valuable model of the inflammatory aspects of multiple sclerosis (MS). Here, we have assessed the role of a σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, in EAE. Experimental Approach EAE was induced in SJL/J female mice by active immunization with myelin proteolipid protein (PLP)139–151 peptide. The σ1 protein agonist was injected i.p. at the time of immunization (day 0). Disease severity was assessed clinically and by histopathological evaluation of the CNS. Phenotyping of B-cell subsets and regulatory T-cells were performed by flow cytometry in spleen and cervical lymph nodes. Key Results Prophylactic treatment of EAE mice with the σ1 protein agonist prevented mononuclear cell accumulation and demyelination in brain and spinal cord and increased T2 B-cells and regulatory T-cells, resulting in an overall reduction in the clinical progression of EAE. Conclusions and Implications This σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, decreased the magnitude of inflammation in EAE. This effect was associated with increased proportions of B-cell subsets and regulatory T-cells with potential immunoregulatory functions. Targeting of the σ1 protein might thus provide new therapeutic opportunities in MS. PMID:25521311

  1. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  2. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-09

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  3. KW-3902, a selective high affinity antagonist for adenosine A1 receptors.

    PubMed Central

    Nonaka, H.; Ichimura, M.; Takeda, M.; Kanda, T.; Shimada, J.; Suzuki, F.; Kase, H.

    1996-01-01

    1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively. PMID:8732272

  4. High affinity nanobodies against the Trypanosome brucei VSG are potent trypanolytic agents that block endocytosis.

    PubMed

    Stijlemans, Benoît; Caljon, Guy; Natesan, Senthil Kumar A; Saerens, Dirk; Conrath, Katja; Pérez-Morga, David; Skepper, Jeremy N; Nikolaou, Alexandros; Brys, Lea; Pays, Etienne; Magez, Stefan; Field, Mark C; De Baetselier, Patrick; Muyldermans, Serge

    2011-06-01

    The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.

  5. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

  6. High-affinity uptake of gamma-aminobutyric acid in cultured glial and neuronal cells.

    PubMed

    Balcar, V J; Mark, J; Borg, J; Mandel, P

    1979-06-01

    Both glial and neuronal cells maintained in primary culture were found to accumulate [3H]GABA by an efficient "high-affinity" uptake system (apparent Km = 9 muM, Vmax = 0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1mM) also strongly inhibited uptake of [3H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, beta-alanine, and 2,4-diaminobutyrate) inhibited uptake of [3H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine, L-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (Km = 92 muM, Vmax = 0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [3H]GABA against a concentration gradient. Apparent Km of this uptake was relatively high (819 muM), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, non of the nontransformed continuous cell lines of either tumoral (glioma, C6; neuroblastoma, M1; M1NN) or normal (NN;I6) origin actively accumulated [3H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.

  7. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors

    PubMed Central

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V.; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging. PMID:27199738

  8. Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

    SciTech Connect

    Lanier, S.M.; Hess, H.J.; Grodski, A.; Graham, R.M.; Homcy, C.J.

    1986-03-01

    The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-phenethyl)carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-(N-4-aminophenethyl)carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand (3H)rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC (17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -(125I)iodophenethyl)carboxamide), was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM).

  9. Sugar-binding proteins from fish: selection of high affinity "lambodies" that recognize biomedically relevant glycans.

    PubMed

    Hong, Xia; Ma, Mark Z; Gildersleeve, Jeffrey C; Chowdhury, Sudipa; Barchi, Joseph J; Mariuzza, Roy A; Murphy, Michael B; Mao, Li; Pancer, Zeev

    2013-01-18

    Glycan-binding proteins are important for a wide variety of basic research and clinical applications, but proteins with high affinity and selectivity for carbohydrates are difficult to obtain. Here we describe a facile and cost-effective strategy to generate monoclonal lamprey antibodies, called lambodies, that target glycan determinants. We screened a library of yeast surface-displayed (YSD) lamprey variable lymphocyte receptors (VLR) for clones that can selectively bind various biomedically important glycotopes. These glycoconjugates included tumor-associated carbohydrate antigens (Tn and TFα), Lewis antigens (LeA and LeX), N-glycolylneuraminic acid, targets of broadly neutralizing HIV antibodies (poly-Man9 and the HIV gp120), and the glycoproteins asialo-ovine submaxillary mucin (aOSM) and asialo-human glycophorin A (aGPA). We isolated clones that bind each of these targets in a glycan-dependent manner and with very strong binding constants, for example, 6.2 nM for Man9 and 44.7 nM for gp120, determined by surface plasmon resonance (SPR). One particular lambody, VLRB.aGPA.23, was shown by glycan array analysis to be selective for the blood group H type 3 trisaccharide (BG-H3, Fucα1-2Galβ1-3GalNAcα), aGPA, and TFα (Galβ1-3GalNAcα), with affinity constants of 0.2, 1, and 8 nM, respectively. In human tissue microarrays this lambody selectively detected cancer-associated carbohydrate antigens in 14 different types of cancers. It stained 27% of non-small cell lung cancer (NSCLC) samples in a pattern that correlated with poor patient survival. Lambodies with exquisite affinity and selectivity for glycans may find myriad uses in glycobiology and biomedical research.

  10. High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

    SciTech Connect

    Nielsen, F.C.; Gammeltoft, S.; Westermark, B.; Fahrenkrug, J. )

    1990-11-01

    Vasoactive intestinal peptide (VIP) bound with high affinity (Kd 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin, PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and (des-His1)VIP bound with 10 and 100 times lower affinity. The fragment VIP(7-28) displaced 25% of the receptor-bound {sup 125}I-VIP whereas VIP(16-28) and VIP(1-22-NH2) were inactive. The binding of {sup 125}I-VIP could be completely inhibited by 10 mumol/l of the antagonists (N-Ac-Tyr1,D-Phe2)GRF(1-29)-NH2, (pCl-D-Phe6,Leu17)VIP and VIP(10-28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated {sup 125}I-VIP was bound to receptors on the cell surface. The internalized {sup 125}I-VIP was completely degraded to {sup 125}I-tyrosine which was released from the cells. Degradation of internalized {sup 125}I-VIP was significantly reduced by chloroquine phenanthroline and pepstatin-A. Surface binding and internalization of {sup 125}I-VIP was increased 3 times by phenanthroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound {sup 125}I-VIP, but caused retention of internalized {sup 125}I-VIP.

  11. Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells.

    PubMed

    Biesbroeck, R; Oram, J F; Albers, J J; Bierman, E L

    1983-03-01

    Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.

  12. Biochemical characterization of high-affinity 3H-opioid binding. Further evidence for Mu1 sites

    SciTech Connect

    Nishimura, S.L.; Recht, L.D.; Pasternak, G.W.

    1984-01-01

    In saturation studies with (/sup 3/H)dihydromorphine, unlabeled D-Ala2-D-Leu5-enkephalin (1 nM) inhibited the high-affinity binding component far more potently than the lower-affinity one. Similarly, morphine (1 nM) inhibited the higher-affinity binding of /sup 3/H-D-Ala2-D-Leu5-enkephalin to a greater extent than its lower-affinity binding component, consistent with a common high-affinity binding site for opiates and enkephalins. Treatment of tissue with either trypsin (1 microgram/ml) or N-ethylmaleimide (25 microM) effectively eliminated the high-affinity binding component of a series of /sup 3/H-opiates and opioid peptides. Competition studies following both treatments were consistent with a common high-affinity binding site. Both treatments also eliminated the ability of low morphine concentrations (less than 1 nM) to inhibit /sup 3/H-D-Ala2-D-Leu5-enkephalin binding and of low D-Ala2-D-Leu5-enkephalin concentrations (less than 1 nM) to inhibit (/sup 3/H)dihydromorphine binding. Protection experiments examining N-ethylmaleimide (25 microM) inhibition of (/sup 3/H)dihydromorphine binding showed significant protection (p less than 0.002) by both unlabeled D-Ala2-D-Leu5-enkephalin and morphine (both at 1 nM). When studied together, both naloxonazine and N-ethylmaleimide inhibited (/sup 3/H)dihydromorphine binding to a similar extent. Equally important, tissue previously treated with naloxonazine was far less sensitive to N-ethylmaleimide than was untreated control tissue, consistent with the possibility that both treatments affected the same site. Together, these results support the concept of a common high-affinity binding site for opiates and opioid peptides.

  13. Sucrose diffusion in aqueous solution

    PubMed Central

    Murray, Benjamin J.

    2016-01-01

    The diffusion of sugar in aqueous solution is important both in nature and in technological applications, yet measurements of diffusion coefficients at low water content are scarce. We report directly measured sucrose diffusion coefficients in aqueous solution. Our technique utilises a Raman isotope tracer method to monitor the diffusion of non-deuterated and deuterated sucrose across a boundary between the two aqueous solutions. At a water activity of 0.4 (equivalent to 90 wt% sucrose) at room temperature, the diffusion coefficient of sucrose was determined to be approximately four orders of magnitude smaller than that of water in the same material. Using literature viscosity data, we show that, although inappropriate for the prediction of water diffusion, the Stokes–Einstein equation works well for predicting sucrose diffusion under the conditions studied. As well as providing information of importance to the fundamental understanding of diffusion in binary solutions, these data have technological, pharmaceutical and medical implications, for example in cryopreservation. Moreover, in the atmosphere, slow organic diffusion may have important implications for aerosol growth, chemistry and evaporation, where processes may be limited by the inability of a molecule to diffuse between the bulk and the surface of a particle. PMID:27364512

  14. Effect of diet on insulin binding and glucose transport in rat sarcolemmal vesicles

    SciTech Connect

    Grimditch, G.K.; Barnard, R.J.; Sternlicht, E.; Whitson, R.H.; Kaplan, S.A.

    1987-03-01

    The purpose of this study was to compare the effects of a high-fat, high-sucrose diet (HFS) and a low-fat, high-complex carbohydrate diet (LFC) on glucose tolerance, insulin binding, and glucose transport in rat skeletal muscle. During the intravenous glucose tolerance test, peak glucose values at 5 min were significantly higher in the HFS group; 0-, 20-, and 60-min values were similar. Insulin values were significantly higher in the HFS group at all time points (except 60 min), indicating whole-body insulin resistance. Skeletal muscle was responsible, in part, for this insulin resistance, because specific D-glucose transport in isolated sarcolemmal (SL) vesicles under basal conditions was similar between LFC and HFS rats, despite the higher plasma insulin levels. Scatchard analyses of insulin binding curves to sarcolemmal vesicles revealed that the K/sub a/ of the high-affinity binding sites was significantly reduced by the HFS diet; no other binding changes were noted. Specific D-glucose transport in SL vesicles after maximum insulin stimulation (1 U/kg) was significantly depressed in the HFS group, indicating that HFS feeding also caused a postbinding defect. These results indicate that the insulin resistance in skeletal muscle associated with a HFS diet is due to both a decrease in the K/sub a/ of the high-affinity insulin receptors and a postbinding defect.

  15. A new therapeutic approach to erectile dysfunction: urotensin-II receptor high affinity agonist ligands.

    PubMed

    di Villa Bianca, Roberta d'Emmanuele; Mitidieri, Emma; Donnarumma, Erminia; Fusco, Ferdinando; Longo, Nicola; Rosa, Giuseppe De; Novellino, Ettore; Grieco, Paolo; Mirone, Vincenzo; Cirino, Giuseppe; Sorrentino, Raffaella

    2015-01-01

    Urotensin-II (U-II) is a cyclic peptide that acts through a G protein-coupled receptor (urotensin-II receptor [UTR]) mainly involved in cardiovascular function in humans. The urotensinergic system is also implicated in the urogenital tract. Indeed, U-II relaxes human corpus cavernosum strips and causes an increase in intracavernous pressure (ICP) in rats. In light of this, the U-II/UTR pathway can be considered a new target for the treatment of erectile dysfunction. On this hypothesis, herein we report on two new UTR high affinity-agonists, P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and UPG84(H-Asp-c[Pen-Phe-DTrp-Orn-(pNH 2 ) Phe-Cys]-Val-OH). The effects of P5U and UPG84 were each compared separately with U-II by monitoring the ICP in anesthetized rats. Intracavernous injection of U-II (0.03-1 nmol), P5U (0.03-1 nmol) or UPG84 (0.03-1 nmol) caused an increase in ICP. P5U, in particular, elicited a significant increase in ICP as compared to U-II. The observed effect by using P5U at a dose of 0.1 nmol per rat was comparable to the effect elicited by U-II at a dose of 0.3 nmol. Moreover, UPG84 at the lowest dose (0.03 nmol) showed an effect similar to the highest dose of U-II (1 nmol). Furthermore, UPG84 was found to be more effective than P5U. Indeed, while the lowest dose of P5U (0.03 nmol) did not affect the ICP, UPG84, at the same dose, induced a prominent penile erection in rat. These compounds did not modify the blood pressure, which indicates a good safety profile. In conclusion, UPG84 and P5U may open new perspectives for the management of erectile dysfunction.

  16. High-affinity VEGF antagonists by oligomerization of a minimal sequence VEGF-binding domain.

    PubMed

    Stefano, James E; Bird, Julie; Kyazike, Josephine; Cheng, Anthony Wai-Ming; Boudanova, Ekaterina; Dwyer, Markryan; Hou, Lihui; Qiu, Huawei; Matthews, Gloria; O'Callaghan, Michael; Pan, Clark Q

    2012-12-19

    Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu(2+) oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ~50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ~2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (~5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc

  17. Developing High-Affinity Protein Capture Agents and Nanotechnology-Based Platforms for In Vitro Diagnostics

    NASA Astrophysics Data System (ADS)

    Rohde, Rosemary Dyane

    In this thesis, I describe projects that were aimed at improving ways to capture proteins for clinical diagnostics. Nanoelectronic sensors, such as silicon nanowires (SiNWs), can provide label-free quantitative measurements of protein biomarkers in real time. One technical challenge for SiNWs is to develop chemistry that can be applied for selectively encoding the nanowire surfaces with capture agents, thus making them sensors that have selectivity for specific proteins. Furthermore, because of the nature of how the sensor works, it is desirable to achieve this spatially selective chemical functionalization without having the silicon undergo oxidation. This method is described here and provides a general platform that can incorporate organic and biological molecules on Si (111) with minimal oxidation of the silicon surface. The development of these devices is, in part, driven by early diagnosis, treatment, monitoring, and personalized medicine---all of which are increasingly requiring quantitative, rapid, and multiparameter measurements. To begin achieving this goal, a large number of protein biomarkers need to be captured and quantitatively measured to create a diagnostic panel. One of the greatest challenges towards making protein-biomarker-based in vitro diagnostics inexpensive involves developing capture agents to detect the proteins. A major thrust of this thesis is to develop multi-valent, high-affinity and high-selectivity protein capture agents using in situ click chemistry. In situ click chemistry is a tool that utilizes the protein itself to catalyze the formation of a biligand from individual azide and alkyne ligands that are co-localized. Large one-bead one-compound (OBOC) libraries of peptides are used to form the body of these ligands, also providing high chemical diversity with minimal synthetic effort. This process can be repeated to identify a triligand, tetraligand, and so forth. Moreover, the resulting multiligand protein capture agents can be

  18. Structural characterization of a high affinity mononuclear site in the copper(II)-α-synuclein complex.

    PubMed

    Bortolus, Marco; Bisaglia, Marco; Zoleo, Alfonso; Fittipaldi, Maria; Benfatto, Maurizio; Bubacco, Luigi; Maniero, Anna Lisa

    2010-12-29

    Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.

  19. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    SciTech Connect

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-03-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.

  20. Identification of a high-affinity ligand that exhibits complete aryl hydrocarbon receptor antagonism.

    PubMed

    Smith, Kayla J; Murray, Iain A; Tanos, Rachel; Tellew, John; Boitano, Anthony E; Bisson, William H; Kolluri, Siva K; Cooke, Michael P; Perdew, Gary H

    2011-07-01

    The biological functions of the aryl hydrocarbon receptor (AHR) can be delineated into dioxin response element (DRE)-dependent or -independent activities. Ligands exhibiting either full or partial agonist activity, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin and α-naphthoflavone, have been demonstrated to potentiate both DRE-dependent and -independent AHR function. In contrast, the recently identified selective AHR modulators (SAhRMs), e.g., 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360), bias AHR toward DRE-independent functionality while displaying antagonism with regard to ligand-induced DRE-dependent transcription. Recent studies have expanded the physiological role of AHR to include modulation of hematopoietic progenitor expansion and immunoregulation. It remains to be established whether such physiological roles are mediated through DRE-dependent or -independent pathways. Here, we present evidence for a third class of AHR ligand, "pure" or complete antagonists with the capacity to suppress both DRE-dependent and -independent AHR functions, which may facilitate dissection of physiological AHR function with regard to DRE or non-DRE-mediated signaling. Competitive ligand binding assays together with in silico modeling identify N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) as a high-affinity AHR ligand. DRE-dependent reporter assays, in conjunction with quantitative polymerase chain reaction analysis of AHR targets, reveal GNF351 as a potent AHR antagonist that demonstrates efficacy in the nanomolar range. Furthermore, unlike many currently used AHR antagonists, e.g., α-naphthoflavone, GNF351 is devoid of partial agonist potential. It is noteworthy that in a model of AHR-mediated DRE-independent function, i.e., suppression of cytokine-induced acute-phase gene expression, GNF351 has the capacity to antagonize agonist and SAhRM-mediated suppression of SAA1. Such data indicate that GNF351 is a

  1. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots1[OPEN

    PubMed Central

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Martínez, Vicente

    2015-01-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K+) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K+ uptake systems in the roots is essential to secure K+ supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K+ concentration is very low (<10 µm), K+ nutrition depends exclusively on the high-affinity K+ transporter5 (HAK5). Low-K+-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca2+ sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K+ transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K+. Experiments of K+ (Rb+) uptake and growth measurements in low-K+ medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta. PMID:26474642

  2. Oligosaccharides from Sucrose via Glycansucrases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glycansucrases are a class of microbial enzymes that polymerize either the fructosyl or the glucosyl moiety of sucrose to give beta-D-fructans or alpha-D-glucans. They are also capable of transferring fructosyl or glucosyl units to acceptor molecules to yield oligosaccharides. Although the glycosy...

  3. Featured Molecules: Sucrose and Vanillin

    NASA Astrophysics Data System (ADS)

    Coleman, William F.; Wildman, Randall J.

    2003-04-01

    The WebWare molecules of the month for April relate to the sense of taste. Apple Fool, the JCE Classroom Activity, mentions sucrose and vanillin and their use as flavorings. Fully manipulable (Chime) versions of these and other molecules are available at Only@JCE Online.

  4. Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation

    PubMed Central

    Goenka, Radhika; Matthews, Andrew H.; Zhang, Bochao; O’Neill, Patrick J.; Scholz, Jean L.; Migone, Thi-Sau; Leonard, Warren J.; Stohl, William; Hershberg, Uri

    2014-01-01

    We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21–mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell–derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence. PMID:24367004

  5. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

  6. Imaging the high-affinity state of the dopamine D2 receptor in vivo: Fact or fiction?

    PubMed Central

    Skinbjerg, Mette; Sibley, David R.; Javitch, Jonathan A.; Abi-Dargham, Anissa

    2013-01-01

    Positron Emission Tomography (PET) has been used for more than three decades to image and quantify dopamine D2 receptors (D2R) in vivo with antagonist radioligands but in the recent years agonist radioligands have also been employed. In vitro competition studies have demonstrated that agonists bind to both a high and a low-affinity state of the D2Rs, of which the high affinity state reflects receptors that are coupled to G-proteins and the low-affinity state reflects receptors uncoupled from G-proteins. In contrast, antagonists bind with uniform affinity to the total pool of receptors. Results of these studies led to the proposal that D2Rs exist in high and low-affinity states for agonists in vivo and sparked the development and use of agonist radioligands for PET imaging with the primary purpose of measuring the proportion of receptors in the high-affinity (activating) state. Although several lines of research support the presence of high and low-affinity states of D2Rs and their detection by in vivo imaging paradigms, a growing body of controversial data has now called this into question. These include both in vivo and ex vivo studies of anesthesia effects, rodent models with increased proportions of high-affinity state D2Rs as well as the molecular evidence for stable receptor–G-protein complexes. In this commentary we review these data and discuss the evidence for the in vivo existence of D2Rs configured in high and low-affinity states and whether or not the high-affinity state of the D2R can, in fact, be imaged in vivo with agonist radioligands. PMID:21945484

  7. High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

    PubMed

    Hara, Yasushi; Tashiro, Yasuyuki; Murakami, Akikazu; Nishimura, Miyuki; Shimizu, Takeyuki; Kubo, Masato; Burrows, Peter D; Azuma, Takachika

    2015-12-01

    During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished.

  8. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    NASA Astrophysics Data System (ADS)

    Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

    2012-03-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  9. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    SciTech Connect

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-05-01

    Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.

  10. Relations between high-affinity binding sites for L-tryptophan, diazepam, salicylate and Phenol Red on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1983-01-01

    Binding of L-tryptophan, diazepam, salicylate and Phenol Red to defatted human serum albumin was studied by ultrafiltration at pH 7.0. All ligands bind to one high-affinity binding site with association constants of the order of 10(4)-10(5)M-1. The number of secondary binding sites was found to vary from zero to five, with association constants about 10(3)M-1. Competitive binding studies with different pairs of the ligands were performed. Binding of both ligands was determined simultaneously. L-Tryptophan and diazepam were found to compete for a common high-affinity binding site on albumin. The following combinations of ligands do not bind competitively to albumin: L-tryptophan-Phenol Red, L-tryptophan-salicylate and Phenol Red-salicylate. On the other hand, high-affinity bindings of the three ligands do not take place independently but in such a way that binding of one of the ligands results in a decrease in binding of the other ligands. The decreases in binding are reciprocal and can be accounted for by introducing a coupling constant. The magnitude of the constant is dependent on the ligands being bound. In the present study, the mutual decrease in binding was more pronounced with L-tryptophan-salicylate and Phenol Red-salicylate than with L-tryptophan-Phenol Red. PMID:6847607

  11. Biosynthesis of sucrose and mannitol as a function of leaf age in celery (Apium graveolens L. )

    SciTech Connect

    Davis, J.M.; Fellman, J.K.; Loescher, W.H.

    1988-01-01

    In celery (Apium graveolens L.), the two major translocated carbohydrates are sucrose and the acyclic polyol mannitol. Their metabolism, however, is different and their specific functions are uncertain. To compare their roles in carbon partitioning and sink-source transitions, developmental changes in /sup 14/CO/sub 2/ labeling, pool sizes, and key enzyme activities in leaf tissues were examined. The proportion of label in mannitol increased dramatically with leaf maturation whereas that in sucrose remained fairly constant. Mannitol content, however, was high in all leaves and sucrose content increased as leaves developed. Activities of mannose-6-P reductase, cytoplasmic and chloroplastic fructose-1,6-bis-phosphatases, sucrose phosphate synthase, and sucrose synthase increased with leaf maturation and decreased as leaves senesced. Ribulose bisphosphate carboxylase and nonreversible glyceraldehyde-3-P dehydrogenase activities rose as leaves developed but did not decrease. Thus, sucrose is produced in all photosynthetically active leaves whereas mannitol is synthesized primarily in mature leaves and stored in all leaves. Onset of sucrose export in celery may result from sucrose accumulation in expanding leaves, but mannitol export is clearly unrelated to mannitol concentration. Mannitol export, however, appears to coincide with increased mannitol biosynthesis. Although mannitol and sucrose arise from a common precursor in celery, subsequent metabolism and transport must be regulated separately.

  12. 21 CFR 172.869 - Sucrose oligoesters.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... mixtures of sucrose fatty acid esters with an average degree of esterification ranging from four to seven. It is produced by interesterification of sucrose with methyl esters of fatty acids derived from... preparation of sucrose oligoesters are dimethyl sulfoxide, isobutyl alcohol, and those solvents...

  13. 21 CFR 172.869 - Sucrose oligoesters.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... mixtures of sucrose fatty acid esters with an average degree of esterification ranging from four to seven. It is produced by interesterification of sucrose with methyl esters of fatty acids derived from... preparation of sucrose oligoesters are dimethyl sulfoxide, isobutyl alcohol, and those solvents...

  14. OCTN1 is a high-affinity carrier of nucleoside analogs.

    PubMed

    Drenberg, Christina; Gibson, Alice A; Pounds, Stanley; Shi, Lei; Rhinehart, Dena; Li, Lie; Hu, Shuiying; Du, Guoqing; Nies, Anne T; Schwab, Matthias; Pabla, Navjotsingh; Blum, William; Gruber, Tanja A; Baker, Sharyn D; Sparreboom, Alex

    2017-02-16

    Resistance to xenobiotic nucleosides used to treat acute myeloid leukemia (AML) and other cancers remains a major obstacle to clinical management. One process suggested to participate in resistance is reduced uptake into tumor cells via nucleoside transporters, although precise mechanisms are not understood. Through transcriptomic profiling, we determined that low expression of the ergothioneine transporter OCTN1 (SLC22A4; ETT) strongly predicts poor event-free survival and overall survival in multiple cohorts of AML patients receiving treatment with the cytidine nucleoside analog cytarabine. Cell biological studies confirmed OCTN1-mediated transport of cytarabine and various structurally-related cytidine analogs, such as 2'deoxycytidine and gemcitabine, occurs through a saturable process that is highly sensitive to inhibition by the classic nucleoside transporter inhibitors dipyridamole and nitrobenzylmercaptopurine ribonucleoside (NBMPR). Our findings have immediate clinical implications given the potential of the identified transport system to help refine strategies that could improve patient survival across multiple cancer types where nucleoside analogs are used in cancer treatment.

  15. Transcriptome Analysis of Sucrose Metabolism during Bulb Swelling and Development in Onion (Allium cepa L.).

    PubMed

    Zhang, Chunsha; Zhang, Hongwei; Zhan, Zongxiang; Liu, Bingjiang; Chen, Zhentai; Liang, Yi

    2016-01-01

    Allium cepa L. is a widely cultivated and economically significant vegetable crop worldwide, with beneficial dietary and health-related properties, but its sucrose metabolism is still poorly understood. To analyze sucrose metabolism during bulb swelling, and the development of sweet taste in onion, a global transcriptome profile of onion bulbs was undertaken at three different developmental stages, using RNA-seq. A total of 79,376 unigenes, with a mean length of 678 bp, was obtained. In total, 7% of annotated Clusters of Orthologous Groups (COG) were involved in carbohydrate transport and metabolism. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, "starch and sucrose metabolism" (147, 2.40%) constituted the primary metabolism pathway in the integrated library. The expression of sucrose transporter genes was greatest during the early-swelling stage, suggesting that sucrose transporters (SUTs) participated in sucrose metabolism mainly at an early stage of bulb development. A gene-expression analysis of the key enzymes of sucrose metabolism suggested that sucrose synthase, cell wall invertase, and invertase were all likely to participate in the hydrolysis of sucrose, generating glucose, and fructose. In addition, trehalose was hydrolyzed to two molecules of glucose by trehalase. From 15 to 40 days after swelling (DAS), both the glucose and fructose contents of bulbs increased, whereas the sucrose content decreased. The growth rate between 15 and 30 DAS was slower than that between 30 and 40 DAS, suggesting that the latter was a period of rapid expansion. The dataset generated by our transcriptome profiling will provide valuable information for further research.

  16. Transcriptome Analysis of Sucrose Metabolism during Bulb Swelling and Development in Onion (Allium cepa L.)

    PubMed Central

    Zhang, Chunsha; Zhang, Hongwei; Zhan, Zongxiang; Liu, Bingjiang; Chen, Zhentai; Liang, Yi

    2016-01-01

    Allium cepa L. is a widely cultivated and economically significant vegetable crop worldwide, with beneficial dietary and health-related properties, but its sucrose metabolism is still poorly understood. To analyze sucrose metabolism during bulb swelling, and the development of sweet taste in onion, a global transcriptome profile of onion bulbs was undertaken at three different developmental stages, using RNA-seq. A total of 79,376 unigenes, with a mean length of 678 bp, was obtained. In total, 7% of annotated Clusters of Orthologous Groups (COG) were involved in carbohydrate transport and metabolism. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, “starch and sucrose metabolism” (147, 2.40%) constituted the primary metabolism pathway in the integrated library. The expression of sucrose transporter genes was greatest during the early-swelling stage, suggesting that sucrose transporters (SUTs) participated in sucrose metabolism mainly at an early stage of bulb development. A gene-expression analysis of the key enzymes of sucrose metabolism suggested that sucrose synthase, cell wall invertase, and invertase were all likely to participate in the hydrolysis of sucrose, generating glucose, and fructose. In addition, trehalose was hydrolyzed to two molecules of glucose by trehalase. From 15 to 40 days after swelling (DAS), both the glucose and fructose contents of bulbs increased, whereas the sucrose content decreased. The growth rate between 15 and 30 DAS was slower than that between 30 and 40 DAS, suggesting that the latter was a period of rapid expansion. The dataset generated by our transcriptome profiling will provide valuable information for further research. PMID:27713754

  17. High-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in neuronal cultures for mechanistic and drug discovery studies.

    PubMed

    Ray, Balmiki; Bailey, Jason A; Simon, Jay R; Lahiri, Debomoy K

    2012-07-01

    Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.

  18. α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

    PubMed

    Absalom, Nathan; Eghorn, Laura F; Villumsen, Inge S; Karim, Nasiara; Bay, Tina; Olsen, Jesper V; Knudsen, Gitte M; Bräuner-Osborne, Hans; Frølund, Bente; Clausen, Rasmus P; Chebib, Mary; Wellendorph, Petrine

    2012-08-14

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA(A) receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA(A) receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC(50) = 140 nM) over α4β(2/3)δ (EC(50) = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA(A) receptors and postulate a role for extrasynaptic α4δ-containing GABA(A) receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

  19. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA.

    PubMed

    Klein, A B; Bay, T; Villumsen, I S; Falk-Petersen, C B; Marek, A; Frølund, B; Clausen, R P; Hansen, H D; Knudsen, G M; Wellendorph, P

    2016-11-01

    GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version ((3)H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations, (3)H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that (3)H-HOCPCA labels only the high-affinity specific GHB binding site, found in high density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd 73.8 nM) compared to pH 7.4 (Kd 2312 nM), as previously reported for other GHB radioligands but similar Bmax values. Using (3)H-HOCPCA we analyzed the GHB binding protein profile during mouse brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that (3)H-HOCPCA is a highly sensitive radioligand, offering advantages over the commonly used radioligand (3)H-NCS-382, and thus a very suitable in vitro tool for qualitative and quantitative autoradiography of the GHB high-affinity site.

  20. Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

    PubMed Central

    Hong, Jung Yeon; Bae, Jong-Hwan; Lee, Kyung Eun; Kim, Mina; Kim, Min Hee; Kang, Hyun Jung; Park, Eun Hye; Yoo, Kyung Sook; Jeong, Se Kyoo; Kim, Kyung Won; Kim, Kyu-Earn

    2016-01-01

    Purpose High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. Materials and Methods Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1atm1Knt Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. Results NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. Conclusion Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases. PMID:27593869

  1. Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

    PubMed

    Nallapareddy, Sreedhar R; Sillanpää, Jouko; Ganesh, Vannakambadi K; Höök, Magnus; Murray, Barbara E

    2007-06-01

    Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P < 0.0001). Blocking Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

  2. Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

    NASA Astrophysics Data System (ADS)

    Steingroewer, Juliane; Bley, Thomas; Bergemann, Christian; Boschke, Elke

    2007-04-01

    Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

  3. Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

    PubMed

    Balashov, Sergei P; Imasheva, Eleonora S; Dioumaev, Andrei K; Wang, Jennifer M; Jung, Kwang-Hwan; Lanyi, Janos K

    2014-12-09

    A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

  4. Affinity labelling and identification of the high-affinity choline carrier from synaptic membranes of Torpedo electromotor nerve terminals with [3H]choline mustard.

    PubMed

    Rylett, R J

    1988-12-01

    The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.

  5. Does the KdpA subunit from the high affinity K(+)-translocating P-type KDP-ATPase have a structure similar to that of K(+) channels?

    PubMed Central

    Durell, S R; Bakker, E P; Guy, H R

    2000-01-01

    Evidence is presented that the transmembrane KdpA subunit of the high affinity K(+)-translocating P-type Kdp-ATPase is evolutionarily derived from the superfamily of 2TM-type K(+) channels in bacteria. This extends a previous study relating the K(+) channels to the KtrAB, Trk, Trk1,2, and HKT1 K(+) symporter superfamily of both prokaryotes and eukaryotes. Although the channels are formed by four single-MPM motif subunits, the transmembrane KdpA subunit and the transmembrane subunit of the symporter proteins are postulated to have four corresponding MPM motifs within a single sequence. Analysis of 17 KdpA sequences reveals a pattern of residue conservation similar to that of the symporters and channels, and consistent with the crystal structure of the KcsA K(+) channel. In addition, the most highly conserved residues between the families, specifically the central glycines of the P2 segments, are those previously identified as crucial for the property of K(+)-selectivity that is common to each protein. This hypothesis is consistent with an experimental study of mutations that alter K(+) binding affinity of the Kdp transporter. Although most of the results of a previous study of the transmembrane topology of KdpA are consistent with the 4-MPM model, the one deviation can be explained by a plausible change in the structure due to the experimental method. PMID:10620285

  6. Breathing air to save energy--new insights into the ecophysiological role of high-affinity [NiFe]-hydrogenase in Streptomyces avermitilis.

    PubMed

    Liot, Quentin; Constant, Philippe

    2016-02-01

    The Streptomyces avermitilis genome encodes a putative high-affinity [NiFe]-hydrogenase conferring the ability to oxidize tropospheric H2 in mature spores. Here, we used a combination of transcriptomic and mutagenesis approaches to shed light on the potential ecophysiological role of the enzyme. First, S. avermitilis was either exposed to low or hydrogenase-saturating levels of H2 to investigate the impact of H2 on spore transcriptome. In total, 1293 genes were differentially expressed, with 1127 and 166 showing lower and higher expression under elevated H2 concentration, respectively. High H2 exposure lowered the expression of the Sec protein secretion pathway and ATP-binding cassette-transporters, with increased expression of genes encoding proteins directing carbon metabolism toward sugar anabolism and lower expression of NADH dehydrogenase in the respiratory chain. Overall, the expression of relA responsible for the synthesis of the pleiotropic alarmone ppGpp decreased upon elevated H2 exposure, which likely explained the reduced expression of antibiotic synthesis and stress response genes. Finally, deletion of hhySL genes resulted in a loss of H2 uptake activity and a dramatic loss of viability in spores. We propose that H2 is restricted to support the seed bank of Streptomyces under a unique survival-mixotrophic energy mode and discuss important ecological implications of this finding.

  7. Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay.

    PubMed

    Zhang, Xian; Sun, Mengjiao; Kang, Yue; Xie, Hui; Wang, Xin; Song, Houhui; Li, Xiaoliang; Fang, Weihuan

    2015-11-01

    Ochratoxin A (OTA) is one of the most commonly occurring mycotoxins produced by some species of Aspergillus and can contaminate cereal and cereal products. A high-affinity anti-OTA monoclonal antibody (mAb) was generated from a hybridoma cell line 2D8 using splenocytes from a BALB/c mouse immunized with synthesized OTA-bovine serum albumin conjugate. The mAb 2D8 is specific with high affinity (3.75 × 10(9) L/M). An indirect competitive ELISA (ic-ELISA) was then developed using this mAb for quantitative determination of OTA in corn and feed samples. Using the optimized conditions, there was good linearity between OTA concentration and competitive inhibition (y = -0.6076x + 0.2441, R(2) = 0.9923) with the working range from 2.4 to 23.6 μg/kg, IC50 at 7.6 μg/kg and lower limit of detection at 1.4 μg/kg. The recovery rates in spiked samples were 91.2-110.3%. Of the 56 corn and feed samples, this ic-ELISA and a commercial kit both found the same 13 samples positive for OTA with good linear correlation between the two methods in OTA quantification (R(2) = 0.9706). We conclude that this ic-ELISA can be used for rapid and quantitative screening of corn and feed samples for the presence of OTA.

  8. Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode.

    PubMed

    Cole, David K; Sami, Malkit; Scott, Daniel R; Rizkallah, Pierre J; Borbulevych, Oleg Y; Todorov, Penio T; Moysey, Ruth K; Jakobsen, Bent K; Boulter, Jonathan M; Baker, Brian M; Yi Li

    2013-01-01

    Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A(∗)0201) complexed with human T cell lymphotropic virus type 111-19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3β loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies.

  9. In vitro toxicological evaluation of NCS-382, a high-affinity antagonist of γ-hydroxybutyrate (GHB) binding.

    PubMed

    Vogel, K R; Ainslie, G R; Roullet, J-B; McConnell, A; Gibson, K M

    2017-01-22

    γ-Hydroxybutyric acid (GHB), a minor metabolite of the inhibitory neurotransmitter GABA, can accumulate to significant concentrations in the heritable disorder of GABA degradation, succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD). Moreover, GHB may be employed in therapeutic settings (treatment of narcolepsy), as well as instances of illicit activity, including acquaintance sexual assault and the induction of euphoria. High-affinity binding sites for GHB in the brain have been identified, although the absolute identity of these receptors remains unclear. Pharmacological antagonism of GHB binding may have multiple instances of therapeutic relevance. The high affinity GHB receptor antagonist, NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid) has not been piloted in humans. To address the potential clinical utility of NCS-382, we have piloted initial studies of its toxicology in HepG2 and primary hepatocyte cells. At high dose (0.5mM), NCS-382 showed no capacity for inhibition of microsomal CYPs (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and minimal potential for activation of xenobiotic nuclear receptors. Additional cellular integrity and functional assays (viability, oxidative stress, apoptosis, ATP production) revealed little evidence for cytotoxicity, and a low degree of dysregulation of >370 genes actively engaged in the mediation of cellular toxicity. In vitro testing indicates a low probability of cellular toxicity associated with NCS-382.

  10. The Structure of the Amyloid-[beta] Peptide High-Affinity Copper II Binding Site in Alzheimer Disease

    SciTech Connect

    Streltsov, Victor A.; Titmuss, Stephen J.; Epa, V. Chandana; Barnham, Kevin J.; Masters, Colin L.; Varghese, Joseph N.

    2008-11-03

    Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-{beta} (A{beta}) protein bound primarily to copper ions. The evidence for an oxidative stress role of A{beta}-Cu redox chemistry is still incomplete. Details of the copper binding site in A{beta} may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of A{beta} peptides complexed with Cu{sup 2+} in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl) concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length A{beta}-Cu{sup 2+} peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the A{beta}-Cu{sup 2+} complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu{sup 2+} binding site is consistent with the hypothesis that the redox activity of the metal ion bound to A{beta} can lead to the formation of dityrosine-linked dimers found in AD.

  11. Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

    PubMed

    Hansson, Karin; Kjellberg, Margareta; Fernlund, Per

    2009-08-01

    BETA-microseminoprotein (MSP), a 10 kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is.

  12. Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.

    PubMed Central

    Pejler, G; David, G

    1987-01-01

    Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed. PMID:2963617

  13. The structure of the amyloid-beta peptide high-affinity copper II binding site in Alzheimer disease.

    PubMed

    Streltsov, Victor A; Titmuss, Stephen J; Epa, V Chandana; Barnham, Kevin J; Masters, Colin L; Varghese, Joseph N

    2008-10-01

    Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-beta (Abeta) protein bound primarily to copper ions. The evidence for an oxidative stress role of Abeta-Cu redox chemistry is still incomplete. Details of the copper binding site in Abeta may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of Abeta peptides complexed with Cu(2+) in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl) concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length Abeta-Cu(2+) peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the Abeta-Cu(2+) complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu(2+) binding site is consistent with the hypothesis that the redox activity of the metal ion bound to Abeta can lead to the formation of dityrosine-linked dimers found in AD.

  14. The Structure of the Amyloid-β Peptide High-Affinity Copper II Binding Site in Alzheimer Disease

    PubMed Central

    Streltsov, Victor A.; Titmuss, Stephen J.; Epa, V. Chandana; Barnham, Kevin J.; Masters, Colin L.; Varghese, Joseph N.

    2008-01-01

    Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-β (Aβ) protein bound primarily to copper ions. The evidence for an oxidative stress role of Aβ-Cu redox chemistry is still incomplete. Details of the copper binding site in Aβ may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of Aβ peptides complexed with Cu2+ in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl) concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length Aβ-Cu2+ peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the Aβ-Cu2+ complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu2+ binding site is consistent with the hypothesis that the redox activity of the metal ion bound to Aβ can lead to the formation of dityrosine-linked dimers found in AD. PMID:18599641

  15. Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3.

    PubMed

    Nilvebrant, Johan; Astrand, Mikael; Löfblom, John; Hober, Sophia

    2013-10-01

    Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.

  16. Biphasic competition between opiates and enkephalins: does it indicate the existence of a common high affinity (mu-1) binding site

    SciTech Connect

    Sarne, Y.; Kenner, A.

    1987-08-03

    Displacement from brain membranes of labeled opiates by low concentrations of enkephalins and of labeled enkephalins by low concentrations of opiates has been previously explained by the existance of a common high affinity site termed mu-1. An alternative interpretation of the same results is that the trough seen in the low concentration zone of the displacement curves represents cross binding of mu and delta opioid ligands to delta and mu receptors, respectively. In three sets of experiments with brain membranes, the size of the trough is shown to be dependent on the labeled ligand used: The ratio between the size of troughs seen with (TH)D-Ala, D-Leu enkephalin and with (TH)morphine varies with experimental conditions (storage of membranes at 4C for 72h), with ratio of mu:delta receptors (e.g. in thalamus and cortex which are enriched in mu and delta sites, respectively) and with pretreatment of membranes with naloxonazine. These results cannot be explained by a common high affinity site, but rather by binding of (TH)D-Ala, D-Leu enkephalin to mu and of (TH)morphine to delta opioid receptors. 17 references, 3 figures.

  17. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy.

    PubMed

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A; Nathenson, Stanley G; Guha, Chandan; Almo, Steven C

    2017-02-06

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  18. Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet.

    PubMed

    Thom, M; Leigh, R A; Maretzki, A

    1986-03-01

    Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[(14)C]glucose in inhibited by high concentrations of osmoticum.

  19. Sucrose synthesis in gamma irradiated sweet potato

    SciTech Connect

    Ailouni, S.; Hamdy, M.K.; Toledo, R.T.

    1987-01-01

    Effect of ..cap alpha..-irradiation carbohydrate metabolism was examined to elucidate mechanism of sucrose accumulation in sweet potato (SP). Enzymes examined were: ..beta..-amylase, phosphorylase, phosphoglucomutase, phosphoglucose isomerase, sucrose phosphate synthetase and sucrose synthetase. Irradiated SP (Red Jewell) sucrose was synthesized to yield 10.7% after 4 d PI. Activities of sugar synthesizing enzymes in irradiated SP were enhanced to different degrees using 100-200 Krad and 3 d PI at 24/sup 0/C. Phosphorylase and phosphoglucomutases specific activities reached 2.4 and 1.8 folds, respectively compared to control SP. ..beta..-amylase, phosphoglucose isomerase, sucrose synthetase and sucrose phosphate synthetase were also affected to yield 1.2, 1.3, 1.3 and 1.2 folds, respectively compared to controls. It is believed that amylase hydrolyzed starch to glucose which is converted to fructose by phosphoglucose isomerase. Sucrose is then formed by sucrose phosphate synthetase and/or sucrose synthetase leading to its accumulation. The irradiated SP was used for alcohol fermentation leading to 500 gal. of 200 proof ethanol/acre (from 500-600 bushels tuber/acre).

  20. Characteristics Of Vacuum Deposited Sucrose Thin Films

    NASA Astrophysics Data System (ADS)

    Ungureanu, F.; Predoi, D.; Ghita, R. V.; Vatasescu-Balcan, R. A.; Costache, M.

    Thin films of sucrose (C12H22O11) were deposited on thin cut glass substrates by thermal evaporation technique (p ~ 10-5 torr). The surface morphology was putted into evidence by FT-IR and SEM analysis. The experimental results confirm a uniform deposition of an adherent sucrose layer. The biological tests (e.g., cell morphology and cell viability evaluated by measuring mitochondrial dehydrogenise activity with MTT assay) confirm the properties of sucrose thin films as bioactive material. The human fetal osteoblast system grown on thin sucrose film was used for the determination of cell proliferation, cell viability and cell morphology studies.

  1. Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum).

    PubMed

    Hajirezaei, Mohammad-Reza; Börnke, Frederik; Peisker, Martin; Takahata, Yasuhiro; Lerchl, Jens; Kirakosyan, Ara; Sonnewald, Uwe

    2003-01-01

    To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased

  2. Neuroimaging of the serotonin reuptake site requires high-affinity ligands.

    PubMed

    Elfving, Betina; Madsen, Jacob; Knudsen, Gitte M

    2007-11-01

    Numerous attempts have been made to develop suitable radiolabeled tracers for positron emission tomography or single photon emission computed tomography imaging of the serotonin transporter (SERT), but most often, negative outcomes are reported. The aim of this study is to define characteristics of a good SERT radioligand and to investigate species differences. We examined seven different selective serotonin reuptake inhibitors (SSRIs) and that except for one all have been previously tested as emission tomography ligands. The outcome of the ligands as emission tomography tracers was compared in relation with receptor density (Bmax) and/or ligand affinity (Kd) in rat and monkey cerebrum and cerebellum (reference region) membranes. [3H]-(S)-Citalopram and [3H]-(+)-McN5652 display statistically significantly lower affinity, whereas [3H]paroxetine displays statistically significantly higher affinity for SERT in monkey cortex when compared with the rat cerebrum. The affinity of [3H]MADAM, [123I]ADAM, and [11C]DASB for SERT obtained with rat cerebrum and monkey cortex are similar. In monkey cortex, Kd and Bmax could not be determined with [3H]fluoxetine. Of the seven SSRIs, [3H]-(S)-citalopram, [3H]MADAM, and [11C]DASB displayed significant specific binding to SERT in monkey cerebellum, with Bmax cortex:cerebellum ratios being 17, 3, and 4, respectively. In rat brain tissue the ratios were 12, 6, and 3, respectively. In conclusion, it can be estimated that imaging of the human SERT in a high-density region requires radioligands with Kd values between 0.03 and a maximum of 0.3 nM (at 37 degrees C). The differential specific cerebellar binding raises the question of the suitability of cerebellum as a reference region for nonspecific binding.

  3. High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking

    PubMed Central

    2010-01-01

    Background STAR/GSG proteins regulate gene expression in metazoans by binding consensus sites in the 5' or 3' UTRs of target mRNA transcripts. Owing to the high degree of homology across the STAR domain, most STAR proteins recognize similar RNA consensus sequences. Previously, the consensus for a number of well-characterized STAR proteins was defined as a hexameric sequence, referred to as the SBE, for STAR protein binding element. C. elegans GLD-1 and mouse Quaking (Qk-1) are two representative STAR proteins that bind similar consensus hexamers, which differ only in the preferred nucleotide identities at certain positions. Earlier reports also identified partial consensus elements located upstream or downstream of a canonical consensus hexamer in target RNAs, although the relative contribution of these sequences to the overall binding energy remains less well understood. Additionally, a recently identified STAR protein called STAR-2 from C. elegans is thought to bind target RNA consensus sites similar to that of GLD-1 and Qk-1. Results Here, a combination of fluorescence-polarization and gel mobility shift assays was used to demonstrate that STAR-2 binds to a similar RNA consensus as GLD-1 and Qk-1. These assays were also used to further delineate the contributions of each hexamer consensus nucleotide to high-affinity binding by GLD-1, Qk-1 and STAR-2 in a variety of RNA contexts. In addition, the effects of inserting additional full or partial consensus elements upstream or downstream of a canonical hexamer in target RNAs were also measured to better define the sequence elements and RNA architecture recognized by different STAR proteins. Conclusions The results presented here indicate that a single hexameric consensus is sufficient for high-affinity RNA binding by STAR proteins, and that upstream or downstream partial consensus elements may alter binding affinities depending on the sequence and spacing. The general requirements determined for high-affinity RNA

  4. In Vivo Analysis of HPr Reveals a Fructose-Specific Phosphotransferase System That Confers High-Affinity Uptake in Streptomyces coelicolor

    PubMed Central

    Nothaft, Harald; Parche, Stephan; Kamionka, Annette; Titgemeyer, Fritz

    2003-01-01

    HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted. The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparent Km of 2 μM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS in S. coelicolor. PMID:12533468

  5. Sucrose accumulation in mature sweet melon fruits. [Cucumis melo

    SciTech Connect

    Schaffer, A.A.; Aloni, B.

    1987-04-01

    Mesocarp tissue from sucrose-accumulating sweet melon (Cucumis melo cv. Galia) showed sucrose synthase activity (ca 1 nkat/gfw) while soluble acid invertase and sucrose phosphate synthase activities were not observed. Sucrose uptake into mesocarp discs was linear with sucrose concentration (1-500 mM) and unaffected by PCMBS and CCCP. Sucrose compartmentation into the vacuole also increased linearly with sucrose concentration as indicated by compartmental efflux kinetics. Mesocarp discs incubated in /sup 14/C-fructose + UDP-glu synthesized /sup 14/C-sucrose and efflux kinetics indicated that the /sup 14/C-sucrose was compartmentalized. These data support the hypothesis that two mechanisms are involved in sucrose accumulation in sweet melon: (1) compartmentation of intact sucrose and (2) synthesis of sucrose via sucrose synthase and subsequent compartmentation in the vacuole.

  6. Sugarcane genes associated with sucrose content

    PubMed Central

    Papini-Terzi, Flávia S; Rocha, Flávia R; Vêncio, Ricardo ZN; Felix, Juliana M; Branco, Diana S; Waclawovsky, Alessandro J; Del Bem, Luiz EV; Lembke, Carolina G; Costa, Maximiller DL; Nishiyama, Milton Y; Vicentini, Renato; Vincentz, Michel GA; Ulian, Eugênio C; Menossi, Marcelo; Souza, Glaucia M

    2009-01-01

    Background - Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. Results - We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. Conclusion - Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be

  7. 21 CFR 172.859 - Sucrose fatty acid esters.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sucrose fatty acid esters. 172.859 Section 172.859... CONSUMPTION Multipurpose Additives § 172.859 Sucrose fatty acid esters. Sucrose fatty acid esters identified...) Sucrose fatty acid esters are the mono-, di-, and tri-esters of sucrose with fatty acids and are...

  8. 21 CFR 172.859 - Sucrose fatty acid esters.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sucrose fatty acid esters. 172.859 Section 172.859... CONSUMPTION Multipurpose Additives § 172.859 Sucrose fatty acid esters. Sucrose fatty acid esters identified...) Sucrose fatty acid esters are the mono-, di-, and tri-esters of sucrose with fatty acids and are...

  9. 21 CFR 172.859 - Sucrose fatty acid esters.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sucrose fatty acid esters. 172.859 Section 172.859... CONSUMPTION Multipurpose Additives § 172.859 Sucrose fatty acid esters. Sucrose fatty acid esters identified...) Sucrose fatty acid esters are the mono-, di-, and tri-esters of sucrose with fatty acids and are...

  10. 21 CFR 172.859 - Sucrose fatty acid esters.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sucrose fatty acid esters. 172.859 Section 172.859... CONSUMPTION Multipurpose Additives § 172.859 Sucrose fatty acid esters. Sucrose fatty acid esters identified...) Sucrose fatty acid esters are the mono-, di-, and tri-esters of sucrose with fatty acids and are...

  11. Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions.

    PubMed

    Himmel, Mirko; Ritter, Anett; Rothemund, Sven; Pauling, Björg V; Rottner, Klemens; Gingras, Alexandre R; Ziegler, Wolfgang H

    2009-05-15

    In cell-extracellular matrix junctions (focal adhesions), the cytoskeletal protein talin is central to the connection of integrins to the actin cytoskeleton. Talin is thought to mediate this connection via its two integrin, (at least) three actin, and several vinculin binding sites. The binding sites are cryptic in the head-to-rod autoinhibited cytoplasmic form of the protein and require (stepwise) conformational activation. This activation process, however, remains poorly understood, and there are contradictory models with respect to the determinants of adhesion site localization. Here, we report turnover rates and protein-protein interactions in a range of talin rod domain constructs varying in helix bundle structure. We conclude that several bundles of the C terminus cooperate to regulate targeting and concomitantly tailor high affinity interactions of the talin rod in cell adhesions. Intrinsic control of ligand binding activities is essential for the coordination of adhesion site function of talin.

  12. Omega-conotoxin GVIA binding to a high-affinity receptor in brain: characterization, calcium sensitivity, and solubilization

    SciTech Connect

    Wagner, J.A.; Snowman, A.M.; Biswas, A.; Olivera, B.M.; Snyder, S.H.

    1988-09-01

    We describe unique, high-affinity binding sites for omega(/sup 125/I)conotoxin GVIA in membranes from rat brain and rabbit sympathetic ganglia which appear to be primarily associated with N-type voltage-dependent calcium channels. The dissociation constant (KD) for the toxin in rat brain membranes is 60 pM. Physiologic extracellular concentrations of calcium inhibit toxin binding noncompetitively (IC50 = 0.2 mM). The regional distribution of the binding sites in rat brain differs markedly from that of dihydropyridine calcium antagonist receptors associated with L-type calcium channels. In detergent-solubilized brain membranes, toxin binding retains the same affinity, specificity, and ionic sensitivity as in particulate preparations.

  13. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

    PubMed Central

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-01-01

    Zr(IV) can form phosphate and Zr(IV) (–PO32−–Zr4+–) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP. PMID:27754349

  14. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    PubMed

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃(2-)-Zr(4+)-) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  15. Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain

    SciTech Connect

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. )

    1991-01-01

    Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.

  16. High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction

    SciTech Connect

    Karatas, Hacer; Townsend, Elizabeth C; Cao, Fang; Chen, Yong; Bernard, Denzil; Liu, Liu; Lei, Ming; Dou, Yali; Wang, Shaomeng

    2013-02-12

    Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein–protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH–, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with Ki < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compound effectively decreases the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein–protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.

  17. Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

    SciTech Connect

    Kim, M.H.; Neubig, R.R.

    1986-03-05

    High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonist (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.

  18. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis

    PubMed Central

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  19. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    PubMed

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction.

  20. High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

    PubMed Central

    Ullman, Christopher; Mathonet, Pascale; Oleksy, Arkadiusz; Diamandakis, Agata; Tomei, Licia; Demartis, Anna; Nardi, Chiara; Sambucini, Sonia; Missineo, Antonino; Alt, Karen; Hagemeyer, Christoph E.; Harris, Matt; Hedt, Amos; Weis, Roland; Gehlsen, Kurt R.

    2015-01-01

    Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model. PMID:26313909

  1. Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.

    PubMed

    Walseth, Timothy F; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S; Slama, James T

    2012-01-20

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.

  2. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

    PubMed

    Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

    2013-12-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

  3. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    SciTech Connect

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-02-10

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 /sup 0/C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17..beta..-(/sup 3/H)estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins.

  4. Histones have high affinity for the glomerular basement membrane. Relevance for immune complex formation in lupus nephritis

    SciTech Connect

    Schmiedeke, T.M.; Stoeckl, F.W.W.; Weber, R.; Sugisaki, Y.; Batsford, S.R.; Vogt, A.

    1989-06-01

    An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns, have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of /sup 125/I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary.

  5. High-Affinity Inhibitors of Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase: Mechanisms of Inhibition and Structure-Activity Relationships

    PubMed Central

    Niesen, Frank H.; Schultz, Lena; Jadhav, Ajit; Bhatia, Chitra; Guo, Kunde; Maloney, David J.; Pilka, Ewa S.; Wang, Minghua; Oppermann, Udo; Heightman, Tom D.; Simeonov, Anton

    2010-01-01

    Background 15-hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies. Principal Findings To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action. Conclusions Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S2. PMID:21072165

  6. 21 CFR 184.1854 - Sucrose.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ....1854 Sucrose. (a) Sucrose (C12H22O11, CAS Reg. No. 57-50-11-1) sugar, cane sugar, or beet sugar is the... sugar beet juice that has been extracted by pressing or diffusion, then clarified and evaporated....

  7. Sucrose- and Fructose-Specific Effects on the Transcriptome of Streptococcus mutans, as Determined by RNA Sequencing.

    PubMed

    Zeng, Lin; Burne, Robert A

    2015-10-16

    Recent genome-scale studies have begun to establish the scope and magnitude of the impacts of carbohydrate source and availability on the regulation of gene expression in bacteria. The effects of sugars on gene expression are particularly profound in a group of lactic acid bacteria that rely almost entirely on their saccharolytic activities for energy production and growth. For Streptococcus mutans, the major etiologic agent of human dental caries, sucrose is the carbohydrate that contributes in the most significant manner to establishment, persistence, and virulence of the organism. However, because this organism produces multiple extracellular sucrolytic enzymes that can release hexoses from sucrose, it has not been possible to study the specific effects of sucrose transport and metabolism on gene expression in the absence of carbohydrates that by themselves can elicit catabolite repression and induce expression of multiple genes. By employing RNA deep-sequencing (RNA-Seq) technology and mutants that lacked particular sucrose-metabolizing enzymes, we compared the transcriptomes of S. mutans bacteria growing on glucose, fructose, or sucrose as the sole carbohydrate source. The results provide a variety of new insights into the impact of sucrose transport and metabolism by S. mutans, including the likely expulsion of fructose after sucrose internalization and hydrolysis, and identify a set of genes that are differentially regulated by sucrose versus fructose. The findings significantly enhance our understanding of the genetics and physiology of this cariogenic pathogen.

  8. Evidence of a calcium-ion-transport system in mitochondria isolated from flight muscle of the developing sheep blowfly Lucilia cuprina.

    PubMed Central

    Bygrave, F L; Daday, A A; Doy, F A

    1975-01-01

    The EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-Ruthenium Red-quench technique (Reed & Bygrave, 1974a) was used to measure initial rates of Ca-2+ transport in mitochondria from flight muscle of the blowfly Lucilia cuprina. Evidence is provided for the existence in these mitochondria of a Ca-2+-transport system that has many features in common with that known to exist in rat liver mitochondria. These include requirement for energy, saturation at high concentrations of Ca-2+, a sigmoidal relation between initial rates of Ca-2+ transport and Ca-2+ concentration, a high affinity for free Ca-2+ (Km approx. 5 muM) and high affinity for the Ca-2+-transport inhibitoy, Ruthenium Red (approx. 0.03 nmol of carrier-specific binding-sites/mg of protein; Ki approx. 1.6 x 10- minus 8 M). Controlled respiration can be stimulated by Ca-2+ after a short lag-period provided the incubation medium contains KCl and not sucrose. The ability of Lucilia mitochondria to transport Ca-2+ critically depends on the stage of mitochondrial development; Ca-2+ transport is minimal in mitochondria from pharate adults, is maximal between 0 and 2h post-emergence and thereafter rapidly declines to reach less than 20% of the maximum value by about 2-3 days post-emergence. Respiration in mitochondria from newly emerged flies does not respond to added Ca-2+; that from 3-5-day-old flies is stimulated approx. 50%. Whereas very low concentrations of Ca-2+ inhibit ADP-stimulated respiration and oxidative phosphorylation in mitochondria from newly emerged flies (Ki approx. 60 ng-ions of Ca-2+/mg of protein); much higher concentrations (approx. 200 ng-ion/mg of protein) are needed to inhibit these processes in those from older flies. The potential of this system for studying the function and development of metabolite transport systems in mitochondria is discussed. PMID:807204

  9. A time-resolved Fourier transformed infrared difference spectroscopy study of the sarcoplasmic reticulum Ca(2+)-ATPase: kinetics of the high-affinity calcium binding at low temperature.

    PubMed Central

    Troullier, A; Gerwert, K; Dupont, Y

    1996-01-01

    We have used time-resolved Fourier transformed infrared difference spectroscopy to characterize the amplitude, frequency, and kinetics of the absorbance changes induced in the infrared (IR) spectrum of sarcoplasmic reticulum Ca(2+)-ATPase by calcium binding at the high-affinity transport sites. 1-(2-Nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis [(oxycarbonyl)methyl]-1,2-ethanediamine (DM-nitrophen) was used as a caged-calcium compound to trigger the release of calcium in the IR samples. Calcium binding to Ca(2+)-ATPase induces the appearance of spectral bands in difference spectra that are all absent in the presence of the inhibitor thapsigargin. Spectral bands above 1700 cm-1 indicate that glutamic and/or aspartic acid side chains are deprotonated upon calcium binding, whereas other bands may be induced by reactions of asparagine, glutamine, and tyrosine residues. Some of the bands appearing in the 1690-1610 cm-1 region arise from modifications of peptide backbone carbonyl groups. The band at 1653 cm-1 is a candidate for a change in an alpha-helix, whereas other bands could arise from modifications of random, turn, or beta-sheet structures or from main-chain carbonyl groups playing the role of calcium ligands. Only a few residues are involved in secondary structure changes. The kinetic evolution of these bands was recorded at low temperature (-9 degrees C). All bands exhibited a monophasic kinetics of rate constant 0.026 s-1, which is compatible with that measured in previous study at the same temperature in a suspension of sarcoplasmic reticulum vesicles by intrinsic fluorescence of Ca(2+)-ATPase. Images FIGURE 4 FIGURE 5 FIGURE 6 PMID:8968569

  10. Sucrose metabolism in lima bean seeds

    SciTech Connect

    Xu, Dianpeng; Sung, Shijean, S.; Black, C.C. )

    1989-04-01

    Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDP-glucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.

  11. Enzymatic conversion of sucrose to hydrogen

    SciTech Connect

    Woodward, J.; Orr, M.

    1998-11-01

    The enzymatic conversion of sugars to hydrogen could be a promising method for alternative fuel production. Maple tree sap is a source of environmental sugar (e.g., sucrose) that has the potential to be converted into hydrogen using the enzymes invertase, glucose dehydrogenase (GDH), hydrogenase, and glucose isomerase (GI) and the cofactor NADP{sup +}/NADPH. The kinetics of hydrogen production have been studied, and optimal conditions for hydrogen production are described. At low initial sucrose concentrations, in the absence of glucose isomerase, stoichiometric yields of mol of H{sub 2}/mol of sucrose were achieved. At higher sucrose concentrations, the yield of hydrogen declined so that at an initial sucrose concentration of 292 mM only 7% yield of hydrogen was obtained. The reason for this low yield was studied and shown not to be caused by enzyme inactivation or a pH drop during the reaction but due to an instability of the cofactor NADP{sup +}. Although gluconic and inhibited both NADPH production and oxidation of GDH and hydrogenase, respectively, it was not the major cause of NADP{sup +} instability. Fructose was also shown to be converted to hydrogen if GI was present in the reaction mixture. Also, by starting with sucrose, 1.34 mol of H{sub 2}/mol of sucrose was obtained if GI was present in the reaction mixture.

  12. Identification of overlapping but differential binding sites for the high-affinity CXCR3 antagonists NBI-74330 and VUF11211.

    PubMed

    Scholten, Danny J; Roumen, Luc; Wijtmans, Maikel; Verkade-Vreeker, Marlies C A; Custers, Hans; Lai, Michael; de Hooge, Daniela; Canals, Meritxell; de Esch, Iwan J P; Smit, Martine J; de Graaf, Chris; Leurs, Rob

    2014-01-01

    CXC chemokine receptor CXCR3 and/or its main three ligands CXCL9, CXCL10, and CXCL11 are highly upregulated in a variety of diseases. As such, considerable efforts have been made to develop small-molecule receptor CXCR3 antagonists, yielding distinct chemical classes of antagonists blocking binding and/or function of CXCR3 chemokines. Although it is suggested that these compounds bind in an allosteric fashion, thus far no evidence has been provided regarding the molecular details of their interaction with CXCR3. Using site-directed mutagenesis complemented with in silico homology modeling, we report the binding modes of two high-affinity CXCR3 antagonists of distinct chemotypes: VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide] (piperazinyl-piperidine) with a rigid elongated structure containing two basic groups and NBI-74330 [(R)-N-(1-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)ethyl)-2-(4-fluoro-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acetamide] (8-azaquinazolinone) without any basic group. Here we show that NBI-74330 is anchored in the transmembrane minor pocket lined by helices 2 (W2.60, D2.63), 3 (F3.32), and 7 (S7.39, Y7.43), whereas VUF11211 extends from the minor pocket into the major pocket of the transmembrane domains, located between residues in helices 1 (Y1.39), 2 (W2.60), 3 (F3.32), 4 (D4.60), 6 (Y6.51), and 7 (S7.39, Y7.43). Mutation of these residues did not affect CXCL11 binding significantly, confirming the allosteric nature of the interaction of these small molecules with CXCR3. Moreover, the model derived from our in silico-guided studies fits well with the already published structure-activity relationship data on these ligands. Altogether, in this study, we show overlapping, yet different binding sites for two high-affinity CXCR3 antagonists, which offer new opportunities for the structure-based design of allosteric modulators for CXCR3.

  13. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    PubMed

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-05

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  14. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    PubMed

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

  15. A Rigid Hinge Region Is Necessary for High-Affinity Binding of Dimannose to Cyanovirin and Associated Constructs.

    PubMed

    Li, Zhen; Bolia, Ashini; Maxwell, Jason D; Bobkov, Andrey A; Ghirlanda, Giovanna; Ozkan, S Banu; Margulis, Claudio J

    2015-11-24

    Mutations in the hinge region of cyanovirin-N (CVN) dictate its preferential oligomerization state. Constructs with the Pro51Gly mutation preferentially exist as monomers, whereas wild-type cyanovirin can form domain-swapped dimers under certain conditions. Because the hinge region is an integral part of the high-affinity binding site of CVN, we investigated whether this mutation affects the shape, flexibility, and binding affinity of domain B for dimannose. Our studies indicate that the capability of monomeric wild-type CVN to resist mechanical perturbations is enhanced when compared to that of constructs in which the hinge region is more flexible. Our computational results also show that enhanced flexibility leads to blocking of the binding site by allowing different rotational isomeric states of Asn53. Moreover, at higher temperatures, this observed flexibility leads to an interaction between Asn53 and Asn42, further hindering access to the binding site. On the basis of these results, we predicted that binding affinity for dimannose would be more favorable for cyanovirin constructs containing a wild-type hinge region, whereas affinity would be impaired in the case of mutants containing Pro51Gly. Experimental characterization by isothermal titration calorimetry of a set of cyanovirin mutants confirms this hypothesis. Those possessing the Pro51Gly mutation are consistently inferior binders.

  16. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    PubMed

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

  17. Receptor regulation of the glutamate, GABA and taurine high-affinity uptake into astrocytes in primary culture.

    PubMed

    Hansson, E; Rönnbäck, L

    1991-05-10

    From experiments using dissociated primary astroglial cultures from newborn rat cerebral cortex, the stimulation of monoamine receptors (alpha, beta and 5HT) was shown to affect the high-affinity uptake kinetics of glutamate, GABA and taurine. In the presence of the alpha 1 agonist phenylephrine, there was an increased uptake (Vmax) of glutamate, while beta adrenoceptor activation slightly inhibited the glutamate uptake and stimulated the GABA and taurine uptakes. 5HT2 receptor stimulation caused a slight inhibition of the taurine uptake. The uptake rate of GABA was not affected by 5HT, alpha 1 or alpha 2 receptor agonists and the glutamate uptake was not affected by 5HT or alpha 2 receptor agonists. Nor was the taurine uptake affected by alpha 1 or alpha 2 receptor agonists. The active uptake of aspartate was unaffected by the presence of any of the monoamine receptor agonists used in this study. When the mechanisms behind these effects were studied, the GABA uptake seemed to be mediated via the G protein-adenylate cyclase complex in the receptor domain. Moreover, the K+ channels seemed to be involved. The taurine uptake, however, did not seem to be regulated by the same mechanism. It seems more probable that there is a direct interaction between the receptor and carrier of taurine at the membrane level. The mechanism underlying the receptor-regulated glutamate uptake is at present unclear, although it does not seem to involve protein kinase C.

  18. High-affinity interactions between peptides and heat shock protein 70 augment CD8+ T lymphocyte immune responses.

    PubMed

    Flechtner, Jessica B; Cohane, Kenya Prince; Mehta, Sunil; Slusarewicz, Paul; Leonard, Alexis Kays; Barber, Brian H; Levey, Daniel L; Andjelic, Sofija

    2006-07-15

    Exogenously delivered antigenic peptides complexed to heat shock proteins (HSPs) are able to enter the endogenous Ag-processing pathway and prime CD8+ CTL. It was determined previously that a hybrid peptide containing a MHC class I-binding epitope and HSP70-binding sequence Javelin (J0) in complex with HSP70 could induce cytotoxic T cell responses in vivo that were more robust than those induced by the minimal epitope complexed with HSP70. The present study introduces a novel, higher-affinity HSP70-binding sequence (J1) that significantly enhances binding of various antigenic peptides to HSP70. A competition binding assay revealed a dissociation constant that was 15-fold lower for the H2-K(b) OVA epitope SIINFEKL-J1 compared with SIINFEKL-J0, indicating a substantially higher affinity for HSP70. Further, modifying the orientation of the hybrid epitope and introducing a cleavable linker sequence between the Javelin and the epitope results in even greater immunogenicity, presumably by greater efficiency of epitope processing. The enhanced immunogenicity associated with Javelin J1 and the cleavable linker is consistently observed with multiple mouse and human epitopes. Thus, by creating a series of epitopes with uniform, high-affinity binding to HSP70, successful multiple epitope immunizations are possible, with equal delivery of each antigenic epitope to the immune system via HSP70. These modified epitopes have the potential for creating successful multivalent vaccines for immunotherapy of both infectious disease and cancer.

  19. Structural basis for pure antagonism of integrin αVβ3 by a high affinity form of fibronectin

    PubMed Central

    Van Agthoven, Johannes F.; Xiong, Jian-Ping; Alonso, José Luis; Rui, Xianliang; Adair, Brian D.; Goodman, Simon L.; Arnaout, M. Amin

    2014-01-01

    Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVβ3 bound to a physiologic ligand: the 10th type III RGD-domain of wild-type fibronectin (wtFN10), or to a high affinity mutant (hFN10) that acts as a pure antagonist. Comparison of these structures revealed a central π - π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the β3-subunit that blocked conformational changes triggered by wtFN10, and trapped hFN10-bound αVβ3 in an inactive conformation. Removing the Trp1496 or Tyr122 side-chains, or reorienting Trp1496 away from Tyr122, converted hFN10 into a partial agonist. The findings offer new insights on the mechanism of integrin activation and a basis for design of RGD-based pure antagonists. PMID:24658351

  20. Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors.

    PubMed Central

    Dionne, C A; Crumley, G; Bellot, F; Kaplow, J M; Searfoss, G; Ruta, M; Burgess, W H; Jaye, M; Schlessinger, J

    1990-01-01

    The fibroblast growth factor (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what factors determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin-like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of 150 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native growth factors, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2-15) x 10(-11) M. The high affinity binding of two distinct growth factors to each of two different receptors represents a unique double redundancy without precedence among polypeptide growth factor-receptor interactions. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1697263

  1. Experimental allergic encephalomyelitis (EAE) in mice selectively bred to produce high affinity (HA) or low affinity (LA) antibody responses.

    PubMed Central

    Devey, M E; Major, P J; Bleasdale-Barr, K M; Holland, G P; Dal Canto, M C; Paterson, P Y

    1990-01-01

    Induction of experimental allergic encephalomyelitis (EAE) in mice genetically selected to produce either high affinity (HA) or low affinity (LA) antibody responses has revealed significant differences in disease susceptibility between the two lines. HA mice were highly susceptible to EAE following subcutaneous sensitization to mouse central nervous system (CNS) tissue emulsified in Freund's complete adjuvant (FCA). Furthermore, of HA mice surviving acute EAE, up to 93% subsequently developed chronic relapsing disease (CREAE) characterized by variable demyelinating inflammatory changes within the spinal cord. In contrast, LA mice, despite having a major histocompatability complex (MHC) haplotype associated with susceptibility to EAE, were highly resistant to the disease and showed no signs of CREAE when observed for up to 100 days post-sensitization. Antibodies to myelin basic protein (MBP) were detected in both lines but rising titres of high functional affinity antibodies were only seen in HA mice. These HA and LA lines of mice provide a new approach to the study of EAE and, in particular, the role of antibody and antibody affinity in the chronic relapsing form of the disease. Images Figure 2 PMID:2335373

  2. A high affinity phage-displayed peptide as a recognition probe for the detection of Salmonella Typhimurium.

    PubMed

    Agrawal, Shailaja; Kulabhusan, Prabir Kumar; Joshi, Manali; Bodas, Dhananjay; Paknikar, Kishore M

    2016-08-10

    Salmonellosis is one of the most common and widely distributed foodborne diseases. A sensitive and robust detection method of Salmonella Typhimurium (S. Typhimurium) in food can critically prevent a disease outbreak. In this work, the use of phage displayed peptides was explored for the detection of S. Typhimurium. A phage-displayed random dodecapeptide library was subjected to biopanning against lipopolysaccharide (LPS) of S. Typhimurium. The peptide NFMESLPRLGMH (pep49) derived from biopanning displayed a high affinity (25.8nM) for the LPS of S. Typhimurium and low cross-reactivity with other strains of Salmonella and related Gram-negative bacteria. Molecular insights into the interaction of pep49 with the LPS of S. Typhimurium was gleaned using atomistic molecular dynamics simulations and docking. It was deduced that the specificity of pep49 with S. Typhimurium LPS originated from the interactions of pep49 with abequose that is found only in the O-antigen of S. Typhimurium. Further, pep49 was able to detect S. Typhimurium at a LOD of 10(3) CFU/mL using ELISA, and may be a potential cost efficient alternative to antibodies.

  3. Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

    PubMed

    Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E; Kozyraki, Renata

    2013-06-07

    Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

  4. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  5. ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

    SciTech Connect

    Sutanto, W.; de Kloet, E.R.

    1988-01-01

    In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15..beta.., 16..beta..b-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of (/sup 3/H) ZK91587 to the total hippocampal coritcosteroid receptor sites with high affinity, and low capacity. When 100-fold excess RU28362 was included simultaneously with (/sup 3/H) ZK91587, the labelled steroid binds with the same affinity and capacity. Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) > cortisol (F); Type II: B > F >>> ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat. The steroid dissociates following a one slope pattern, indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like receptor.

  6. The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

    PubMed

    Chenoweth, Alicia M; Trist, Halina M; Tan, Peck-Szee; Wines, Bruce D; Hogarth, P Mark

    2015-11-01

    Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

  7. High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

    PubMed

    Goldman, Johnathan M; Zhang, Li Ang; Manna, Arunava; Armitage, Bruce A; Ly, Danith H; Schneider, James W

    2013-07-08

    Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

  8. Development of indazolylpyrimidine derivatives as high-affine EphB4 receptor ligands and potential PET radiotracers.

    PubMed

    Ebert, Kristin; Wiemer, Jens; Caballero, Julio; Köckerling, Martin; Steinbach, Jörg; Pietzsch, Jens; Mamat, Constantin

    2015-09-01

    Due to their essential role in the pathogenesis of cancer, members of the Eph (erythropoietin-producing hepatoma cell line-A2) receptor tyrosine kinase family represent promising candidates for molecular imaging. Thus, the development and preparation of novel radiotracers for the noninvasive imaging of the EphB4 receptor via positron emission tomography (PET) is described. First in silico investigations with the indazolylpyrimidine lead compound which is known to be highly affine to EphB4 were executed to identify favorable labeling positions for an introduction of fluorine-18 to retain the affinity. Based on this, reference compounds as well as precursors were developed and labeled with carbon-11 and fluorine-18, respectively. For this purpose, a protecting group strategy essentially had to be generated to prevent unwanted methylation and to enable the introduction of fluorine-18. Further, a convenient radiolabeling strategy using [(11)C]methyl iodide was established which afforded the isotopically labeled radiotracer in 30-35% RCY (d.c.) which is identical with the original inhibitor molecule. A spiro ammonium precursor was prepared for radiolabeling with fluorine-18. Unfortunately, the labeling did not lead to the desired (18)F-radiotracer under the chosen conditions.

  9. A Soluble Form of the High Affinity IgE Receptor, Fc-Epsilon-RI, Circulates in Human Serum

    PubMed Central

    Dehlink, Eleonora; Platzer, Barbara; Baker, Alexandra H.; LaRosa, Jessica; Pardo, Michael; Dwyer, Peter; Yen, Elizabeth H.; Szépfalusi, Zsolt

    2011-01-01

    Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI), the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum. PMID:21544204

  10. Synthesis of tetravalent LacNAc-glycoclusters as high-affinity cross-linker against Erythrina cristagalli agglutinin.

    PubMed

    Ogata, Makoto; Chuma, Yasushi; Yasumoto, Yoshinori; Onoda, Takashi; Umemura, Myco; Usui, Taichi; Park, Enoch Y

    2016-01-01

    Four kinds of tetravalent double-headed glycoclusters [(LacNAc)4-DHGs] were designed with linkers of varying lengths consisting of alkanedioic carboxyamido groups (C6, C12, C18 and C24) between two bi-antennary LacNAc-glycosides. These glycoclusters served as high-affinity cross-linking ligands for the LacNAc-binding lectin Erythrina cristagalli agglutinin (ECA). The binding activity and cross-linking between each ligand and ECA were characterized by a hemagglutination inhibition (HI) assay, isothermal titration calorimetry (ITC), a quantitative precipitation assay and dynamic light scattering (DLS). For the precipitation assay and DLS measurement, the synthesized (LacNAc)4-DHGs were found to be capable of binding and precipitating the ECA as multivalent ligands. ITC analysis indicated the binding of (LacNAc)4-DHGs was driven by a favorable enthalpy change. Furthermore, the entropy penalty from binding (LacNAc)4-DHGs clearly decreased in a spacer length-dependent manner. The binding affinities of flexible (LacNAc)4-DHGs (C18 and C24) with long spacers were found to be more favorable than those of the clusters having short spacers (C6 and C12). These results were supported by molecular dynamics simulations with explicit water molecules for the tetravalent glycoclusters with ECA. We concluded that the subtle modification in the epitope-presenting scaffolds exerts the significant effect in the recognition efficiency involved in the LacNAc moieties by ECA.

  11. The Analysis of the Human High Affinity IgE Receptor FceRIa from Multiple Crystal Forms

    SciTech Connect

    Garman, S.C.; Sechi, S.; Kinet, J.-P.; Jardetzky, T.S.

    2010-03-05

    We have solved the structure of the human high affinity IgE receptor, Fc{var_epsilon}RI{alpha}, in six different crystal forms, showing the structure in 15 different chemical environments. This database of structures shows no change in the overall shape of the molecule, as the angle between domains 1 and 2 (D1 and D2) varies little across the ensemble. However, the receptor has local conformational variability in the C' strand of D2 and in the BC loop of D1. In every crystal form, a residue inserts between tryptophan residues 87 and 110, mimicking the position of a proline from the IgE ligand. The different crystal forms reveal a distribution of carbohydrates lining the front and back surfaces of the structure. An analysis of crystal contacts in the different forms indicates regions where the molecule interacts with other proteins, and reveals a potential new binding site distal to the IgE binding site. The results of this study point to new directions for the design of molecules to inhibit the interaction of Fc{var_epsilon}RI{alpha} with its natural ligand and thus to prevent a primary step in the allergic response.

  12. Identification of a high-affinity binding site for dinotefuran in the nerve cord of the American cockroach.

    PubMed

    Miyagi, Satoshi; Komaki, Iori; Ozoe, Yoshihisa

    2006-04-01

    The binding of the neonicotinoid insecticide dinotefuran to insect nicotinic acetylcholine receptors (nAChRs) was examined by a centrifugation method using the nerve cord membranes of American cockroaches and [3H]dinotefuran (78 Ci mmol-1). The Kd and Bmax values of [3H]dinotefuran binding were estimated to be 13.7 nM and 14.8 fmol 40 microg-1 protein respectively by Scatchard analysis. Epibatidine, an nAChR agonist, showed a rather lower affinity to the dinotefuran binding site (IC50=991 nM) than dinotefuran (IC50=5.02 nM). Imidacloprid and nereistoxin displayed lower potencies than dinotefuran but higher potencies than epibatidine. The potencies of five dinotefuran analogues in inhibiting the specific binding of [3H]dinotefuran to nerve cord membranes were determined. A good correlation (r2=0.970) was observed between the -log IC50 values of the tested compounds and their piperonyl butoxide-synergised insecticidal activities (-log LD50 values) against German cockroaches. The results indicate that a high-affinity binding site for dinotefuran is present in the nerve cord of the American cockroach and that the binding of ligands to the site leads to the manifestation of insecticidal activity.

  13. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    SciTech Connect

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. )

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  14. Allosteric Inhibition of a Semaphorin 4D Receptor Plexin B1 by a High-Affinity Macrocyclic Peptide.

    PubMed

    Matsunaga, Yukiko; Bashiruddin, Nasir K; Kitago, Yu; Takagi, Junichi; Suga, Hiroaki

    2016-11-17

    Semaphorin axonal guidance factors are multifunctional proteins that play important roles in immune response, cancer cell proliferation, and organogenesis, making semaphorins and their signaling receptor plexins important drug targets for various diseases. However, the large and flat binding surface of the semaphorin-plexin interaction interface is difficult to target by traditional small-molecule drugs. Here, we report the discovery of a high-affinity plexin B1 (PlxnB1)-binding macrocyclic peptide, PB1m6 (KD = 3.5 nM). PB1m6 specifically inhibited the binding of physiological ligand semaphorin 4D (Sema4D) in vitro and completely suppressed Sema4D-induced cell collapse. Structural studies revealed that PB1m6 binds at a groove between the fifth and sixth blades of the sema domain in PlxnB1 distant from the Sema4D-binding site, indicating the non-competitive and allosteric nature of the inhibitory activity. The discovery of this novel allosteric site can potentially be used to target plexin family proteins for the development of drugs that modulate semaphorin and plexin signaling.

  15. G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

    PubMed Central

    Tatsumi, Kasumi; Sakashita, Gyosuke; Nariai, Yuko; Okazaki, Kosuke; Kato, Hiroaki; Obayashi, Eiji; Yoshida, Hisashi; Sugiyama, Kanako; Park, Sam-Yong; Sekine, Joji; Urano, Takeshi

    2017-01-01

    The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research. PMID:28266535

  16. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  17. Surprisingly High Selectivity and High Affinity in Mercury Recognition by H-Bonded Cavity-Containing Aromatic Foldarands.

    PubMed

    Shen, Jie; Ren, Changliang; Zeng, Huaqiang

    2017-02-09

    In the absence of macrocyclic ring constraints, few synthetic systems, possessing a mostly solvent-independent well-folded conformation that is predisposed for highly selective and high affinity recognition of metal ions, have been demonstrated. We report here such a unique class of conformationally robust modularly tunable folding molecules termed foldarands that can recognize Hg(2+) ions surprisingly well over 22 other metal ions. Despite the lack of sulfur atoms and having only oxygen-donor atoms in its structure, the best foldarand molecule, i.e., tetramer 4, exhibits a selectivity factor of at least 19 in differentiating the most tightly bound Hg(2+) ion from all other metal ions, and a binding capacity that is ≥18 times that of thio-crown ethers. These two noteworthy binding characters make possible low level removal of Hg(2+) ions. With a [4]:[Hg(2+)] molar ratio of 5:1 and a single biphasic solvent extraction, the concentration of Hg(2+) ions could be reduced drastically by 98% (from 200 to 4 ppb) in pure water. 4 could also effect a highly efficient reduction in mercury content by 98% (from 500 to 10 ppb) in artificial groundwater via multiple successive extractions with an overall consumption of 4 being 9:1 in terms of [4]:[Hg(2+)] molar ratio.

  18. OB-fold domain of KREPA4 mediates high-affinity interaction with guide RNA and possesses annealing activity.

    PubMed

    Kala, Smriti; Salavati, Reza

    2010-10-01

    KREPA4, also called MP24, is an essential mitochondrial guide RNA (gRNA)-binding protein with a preference for the 3' oligo(U) tail in trypanosomes. Structural prediction and compositional analysis of KREPA4 have identified a conserved OB (oligonucleotide/oligosaccharide-binding)-fold at the C-terminal end and two low compositional complexity regions (LCRs) at its N terminus. Concurrent with these predictions, one or both of these regions in KREPA4 protein may be involved in gRNA binding. To test this possibility, deletion mutants of KREPA4 were made and the effects on the gRNA-binding affinities were measured by quantitative electrophoretic mobility shift assays. The gRNA-binding specificities of these mutants were evaluated by competition experiments using gRNAs with U-tail deletions or stem-loop modifications and uridylated nonguide RNAs or heterologous RNA. Our results identified the predicted OB-fold as the functional domain of KREPA4 that mediates a high-affinity interaction with the gRNA oligo(U) tail. An additional contribution toward RNA-binding function was localized to LCRs that further stabilize the binding through sequence-specific interactions with the guide secondary structure. In this study we also found that the predicted OB-fold has an RNA annealing activity, representing the first report of such activity for a core component of the RNA editing complex.

  19. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

    PubMed

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.

  20. New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

    PubMed

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-05-21

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

  1. Cubilin, a High Affinity Receptor for Fibroblast Growth Factor 8, Is Required for Cell Survival in the Developing Vertebrate Head*

    PubMed Central

    Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P.; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E.; Kozyraki, Renata

    2013-01-01

    Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity. PMID:23592779

  2. Temperature-sensitive high affinity (/sup 3/H)serotonin binding: characterization and effects of antidepressant treatment

    SciTech Connect

    Helmeste, D.M.; Tang, S.W.

    1984-08-13

    Characterization of temperature-sensitive (/sup 3/H)serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S/sub 1/ and S/sub 2/ receptors. In vivo pretreatment (48 h before) with mianserin did not alter B/sub max/ or Kd for the 1 nM Kd (/sup 3/H)5-HT site, although (/sup 3/H)ketanserin (S/sub 2/) densities were decreased by 50%. This suggested that possible S/sub 2/ components of (/sup 3/H)5-HT binding must be negligible, even though ketanserin competed with high affinity (IC/sub 50/ = 3 nM) for a portion of the 1 nM Kd (/sup 3/H)5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd (/sup 3/H)5-HT site in a non-competitive manner, as shown by a decrease in B/sub max/ with no change in Kd after in vitro incubation. The complex inhibition data may therefore represent indirect interactions through another site.

  3. Are high-affinity progesterone binding site(s) from porcine liver microsomes members of the sigma receptor family?

    PubMed

    Meyer, C; Schmieding, K; Falkenstein, E; Wehling, M

    1998-04-24

    Membrane progesterone binding sites have been purified recently from pig liver. Since progesterone is considered as an endogenous sigma (sigma) receptor ligand, these sites were characterized pharmacologically by ligands selective for sigma receptor and dopamine receptor binding sites, and by other drugs from distinct pharmacological classes. Binding studies using the radioligand [3H]progesterone were done in crude membrane preparations and solubilized fractions to determine half-maximal inhibitory concentration (IC50) values, from which inhibitory constants (Ki values) were calculated. Radioligand binding was inhibited by the sigma receptor ligands haloperidol, carbetapentane citrate, 1,3-Di(2-tolyl)guanidine (DTG), R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2 aminopropane HCl (R(-)-PPAAP HCl), or sigma receptor antagonists like (+)-3-(3-hydroxyphenyl)-N-propylpiperidine HCl (R(+)-PPP HCl) and cis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]-9H-carbazole dihydrochloride (rimcazole 2HCl). The hierarchy of inhibitory action was not fully compatible with either sigma receptor class I (moderate affinity of pentazocine, diphenylhydantoin (phenytoin) insensitivity) or II sites (high affinity of carbetapentane). The data thus suggest that progesterone binding sites in porcine liver membranes are related to the sigma receptor binding site superfamily, but may represent a particular species with progesterone specificity.

  4. 21 CFR 184.1854 - Sucrose.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... sugar, or beet sugar is the chemical β-D-fructofuranosyl-α-D-glucopyranoside. Sucrose is obtained by crystallization from sugar cane or sugar beet juice that has been extracted by pressing or diffusion,...

  5. 21 CFR 184.1854 - Sucrose.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... sugar, or beet sugar is the chemical β-D-fructofuranosyl-α-D-glucopyranoside. Sucrose is obtained by crystallization from sugar cane or sugar beet juice that has been extracted by pressing or diffusion,...

  6. 21 CFR 184.1854 - Sucrose.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... sugar, or beet sugar is the chemical β-D-fructofuranosyl-α-D-glucopyranoside. Sucrose is obtained by crystallization from sugar cane or sugar beet juice that has been extracted by pressing or diffusion,...

  7. 21 CFR 184.1854 - Sucrose.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... sugar, or beet sugar is the chemical β-D-fructofuranosyl-α-D-glucopyranoside. Sucrose is obtained by crystallization from sugar cane or sugar beet juice that has been extracted by pressing or diffusion,...

  8. 21 CFR 172.869 - Sucrose oligoesters.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dimethyl sulfoxide, isobutyl alcohol, and those solvents generally recognized as safe in food. (b) Sucrose... Convention, 12601 Twinbrook Pkwy., Rockville, MD 20852 (Internet address http://www.usp.org) (7) Residue...

  9. Structural development of sucrose-sweetened and sucrose-free sponge cakes during baking.

    PubMed

    Baeva, Marianna Rousseva; Terzieva, Vesselina Velichkova; Panchev, Ivan Nedelchev

    2003-06-01

    The influence of sucrose, wheat starch and sorbitol upon the heat- and mass-exchanging processes forming the structure of sponge cake was studied. Under the influence of wheat starch and sorbitol the structure of the sucrose-free sponge cake was formed at more uniform total moisture release. This process was done at lower temperatures and smoother change of the sponge cake height with respect to the sucrose-sweetened sponge cake. The porous and steady structure of both cakes was finally formed at identical time--between 18th and 19th minute, at the applied conditions for baking of each batter (metal pan with diameter 15.4 cm and depth 6.2 cm containing 300 g of batter and placed in an electric oven "Rahovetz-02", Bulgaria for 30 min at 180 degrees C). The water-losses at the end of baking (10.30% and 10.40% for the sucrose-sweetened cake and sucrose-free cake, respectively) and the final temperatures reached in the crumb central layers (96.6 degrees C and 96.3 degrees C for the sucrose-sweetened cake and sucrose-free cake, respectively) during baking of both samples were not statistically different. The addition of wheat starch and sorbitol in sucrose-free sponge cake lead to the statistically different values for the porosity (76.15% and 72.98%) and the volume (1014.17 cm3 and 984.25 cm3) of the sucrose-sweetened and sucrose-free sponge cakes, respectively. As a result, the sucrose-free sponge cake formed during baking had a more homogeneous and finer microstructure with respect to that ofthe sucrose-sweetened one.

  10. The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity.

    PubMed

    Verhaeghe, Tom; Aerts, Dirk; Diricks, Margo; Soetaert, Wim; Desmet, Tom

    2014-08-01

    Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis.

  11. Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data.

    PubMed Central

    Rohwer, J M; Botha, F C

    2001-01-01

    Sucrose accumulation in developing sugar cane (Saccharum officinarum) is accompanied by a continuous synthesis and cleavage of sucrose in the storage tissues. Despite numerous studies, the factors affecting sucrose accumulation are still poorly understood, and no consistent pattern has emerged which pinpoints certain enzyme activities as important controlling steps. Here, we develop an approach based on pathway analysis and kinetic modelling to assess the biochemical control of sucrose accumulation and futile cycling in sugar cane. By using the concept of elementary flux modes, all possible routes of futile cycling of sucrose were enumerated in the metabolic system. The available kinetic data for the pathway enzymes were then collected and assembled in a kinetic model of sucrose accumulation in sugar cane culm tissue. Although no data were fitted, the model agreed well with independent experimental results: in no case was the difference between calculated and measured fluxes and concentrations greater than 2-fold. The model thus validated was then used to assess different enhancement strategies for increasing sucrose accumulation. First, the control coefficient of each enzyme in the system on futile cycling of sucrose was calculated. Secondly, the activities of those enzymes with the numerically largest control coefficients were varied over a 5-fold range to determine the effect on the degree of futile cycling, the conversion efficiency from hexoses into sucrose, and the net sucrose accumulation rate. In view of the modelling results, overexpression of the fructose or glucose transporter or the vacuolar sucrose import protein, as well as reduction of cytosolic neutral invertase levels, appear to be the most promising targets for genetic manipulation. This offers a more directed improvement strategy than cumbersome gene-by-gene manipulation. The kinetic model can be viewed and interrogated on the World Wide Web at http://jjj.biochem.sun.ac.za. PMID:11513743

  12. Down-Regulating CsHT1, a Cucumber Pollen-Specific Hexose Transporter, Inhibits Pollen Germination, Tube Growth, and Seed Development1[OPEN

    PubMed Central

    Cheng, Jintao; Wang, Zhenyu; Yao, Fengzhen; Gao, Lihong; Ma, Si; Zhang, Zhenxian

    2015-01-01

    Efficient sugar transport is needed to support the high metabolic activity of pollen tubes as they grow through the pistil. Failure of transport results in male sterility. Although sucrose transporters have been shown to play a role in pollen tube development, the role of hexoses and hexose transporters is not as well established. The pollen of some species can grow in vitro on hexose as well as on sucrose, but knockouts of individual hexose transporters have not been shown to impair fertilization, possibly due to transporter redundancy. Here, the functions of CsHT1, a hexose transporter from cucumber (Cucumis sativus), are studied using a combination of heterologous expression in yeast (Saccharomyces cerevisiae), histochemical and immunohistochemical localization, and reverse genetics. The results indicate that CsHT1 is a plasma membrane-localized hexose transporter with high affinity for glucose, exclusively transcribed in pollen development and expressed both at the levels of transcription and translation during pollen grain germination and pollen tube growth. Overexpression of CsHT1 in cucumber pollen results in a higher pollen germination ratio and longer pollen tube growth than wild-type pollen in glucose- or galactose-containing medium. By contrast, antisense suppression of CsHT1 leads to inhibition of pollen germination and pollen tube elongation in the same medium and results in a decrease of seed number per fruit and seed size when antisense transgenic pollen is used to fertilize wild-type or transgenic cucumber plants. The important role of CsHT1 in pollen germination, pollen tube growth, and seed development is discussed. PMID:25888616

  13. Down-Regulating CsHT1, a Cucumber Pollen-Specific Hexose Transporter, Inhibits Pollen Germination, Tube Growth, and Seed Development.

    PubMed

    Cheng, Jintao; Wang, Zhenyu; Yao, Fengzhen; Gao, Lihong; Ma, Si; Sui, Xiaolei; Zhang, Zhenxian

    2015-06-01

    Efficient sugar transport is needed to support the high metabolic activity of pollen tubes as they grow through the pistil. Failure of transport results in male sterility. Although sucrose transporters have been shown to play a role in pollen tube development, the role of hexoses and hexose transporters is not as well established. The pollen of some species can grow in vitro on hexose as well as on sucrose, but knockouts of individual hexose transporters have not been shown to impair fertilization, possibly due to transporter redundancy. Here, the functions of CsHT1, a hexose transporter from cucumber (Cucumis sativus), are studied using a combination of heterologous expression in yeast (Saccharomyces cerevisiae), histochemical and immunohistochemical localization, and reverse genetics. The results indicate that CsHT1 is a plasma membrane-localized hexose transporter with high affinity for glucose, exclusively transcribed in pollen development and expressed both at the levels of transcription and translation during pollen grain germination and pollen tube growth. Overexpression of CsHT1 in cucumber pollen results in a higher pollen germination ratio and longer pollen tube growth than wild-type pollen in glucose- or galactose-containing medium. By contrast, antisense suppression of CsHT1 leads to inhibition of pollen germination and pollen tube elongation in the same medium and results in a decrease of seed number per fruit and seed size when antisense transgenic pollen is used to fertilize wild-type or transgenic cucumber plants. The important role of CsHT1 in pollen germination, pollen tube growth, and seed development is discussed.

  14. 21 CFR 172.859 - Sucrose fatty acid esters.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sucrose fatty acid esters. 172.859 Section 172.859... Sucrose fatty acid esters. Sucrose fatty acid esters identified in this section may be safely used in accordance with the following prescribed conditions: (a) Sucrose fatty acid esters are the mono-, di-,...

  15. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

    PubMed Central

    Levay, Agata; Brenneman, Randall; Hoinka, Jan; Sant, David; Cardone, Marco; Trinchieri, Giorgio; Przytycka, Teresa M.; Berezhnoy, Alexey

    2015-01-01

    Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs. PMID:26007661

  16. Replacement of the Bryostatin A- and B-Pyran Rings With Phenyl Rings Leads to Loss of High Affinity Binding With PKC.

    PubMed

    Petersen, Mark E; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M; Keck, Gary E

    2016-10-19

    We describe a convergent synthesis of a bryostatin analogue in which the natural A- and B-ring pyrans have been replaced by phenyl rings. The new analogue exhibited PMA like behavior in cell assays, but failed to maintain high affinity binding for PKC, despite retaining an unaltered C-ring 'binding domain'.

  17. Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein.

    PubMed Central

    Iwai, S; Pritchard, C; Mann, D A; Karn, J; Gait, M J

    1992-01-01

    The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure. Images PMID:1282702

  18. Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

    PubMed Central

    Shimada, K; Ozawa, T

    1985-01-01

    It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycosaminoglycans such as heparan sulfate, which could serve as antithrombin cofactor on the endothelium. In the present study, we have directly evaluated the binding of 125I-labeled bovine thrombin to cultured porcine aortic endothelial cells. The thrombin binding to the cell surface was rapid, reversible, and displaced by enzymatically inactive diisopropylphosphoryl-thrombin. The concentration of thrombin at half-maximal binding was approximately 20 nM. Both specific and nonspecific binding of 125I-thrombin to the endothelial cell surface was partially inhibited in the presence of protamine sulfate, after the removal of cell surface heparan sulfate by the treatment of cells with crude Flavobacterium heparinum enzyme or purified heparitinase. The binding as a function of the concentration of thrombin revealed that the maximal amount of specific binding was reduced by approximately 50% with little alteration in binding affinity by these enzymatic treatments. The reversibility and active-site independence as well as the rate of the binding did not change after heparitinase treatment. Whereas removal of chondroitin sulfates by chondroitin ABC lyase treatment of cells did not affect the binding, identical enzymatic treatments of [35S]sulfate-labeled cells showed that either heparan sulfate or chondroitin sulfate was selectively and completely removed from the cell surface by heparitinase or chondroitin ABC lyase treatment, respectively. Furthermore, proteolysis of cell surface proteins by the purified glycosaminoglycan lyases was excluded by the identical enzymatic treatments of [3H]leucine-labeled or cell surface radioiodinated cells. Our results provide the first direct evidence that heparan sulfate on the cell surface is involved in the high-affinity, active site-independent thrombin binding by endothelial cells, and also suggest the presence of thrombin

  19. Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

    PubMed

    Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition.

  20. Androgen-induced sexual dimorphism in high affinity dopamine binding in the brain transcends the hypothalamic-limbic region.

    PubMed Central

    Jalilian-Tehrani, M. H.; Karakiulakis, G.; Le Blond, C. B.; Powell, R.; Thomas, P. J.

    1982-01-01

    1 High affinity binding of [3H]-dopamine and [3H]-5-hydroxytryptamine ([3H]-5-HT) was measured in membrane fractions prepared from cerebral cortex, amygdala, hypothalamus, thalamus and brain stem of rats of either sex and of rats which had been either neonatally castrated or androgenized. 2 Binding was measured in rats of 8, 20 and 30 days old as well as in adults. 3 [3H]-dopamine bound with approximately 30 nM affinity ahd [3H]-5-HT with approximately 10 nM affinity to all areas of the brain tested. The relative inhibitory effects of haloperidol, apomorphine, cis-flupenthixol, unlabelled dopamine, noradrenaline, spiroperone, (+)-butaclamol, fluphenazine, pimozide and 5-HT on [3H]-dopamine binding in the cerebral cortex was consistent with receptor status for the binding components there as were the relative inhibitory effects of methysergide, dopamine, fluoxetine and ouabain on [3H]-5-HT binding in the fore brain. 4 Neither [3H]-dopamine nor [3H]-5-HT binding varied with the state of the sexual cycle in females. 5 There were no sexual differences in [3H]-5-HT binding in any of the brain areas tested nor was it affected by neonatal androgenization or neonatal castration. 6 [3H]-dopamine binding was greater in the cerebral cortex and amygdala of male than of female rats. These differences could be mimicked artificially by neonatal castration of males (female type development) or neonatal androgenization of females (male type development). Sexual dimorphism did not become overt until 20 days of age and did not extend to hypothalamus, thalamus or brain stem. 7 It is concluded that neonatal sex differences in exposure to steroid hormones has permanent effects on the number of dopamine binding sites in the cerebral cortex and is suggested that this sexual dimorphism extends to the amygdala. PMID:7074286

  1. Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

    PubMed Central

    Nissim, A; Jouvin, M H; Eshhar, Z

    1991-01-01

    Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI. Images PMID:1824934

  2. Transmembrane-truncated alphavbeta3 integrin retains high affinity for ligand binding: evidence for an 'inside-out' suppressor?

    PubMed Central

    Mehta, R J; Diefenbach, B; Brown, A; Cullen, E; Jonczyk, A; Güssow, D; Luckenbach, G A; Goodman, S L

    1998-01-01

    The molecular mechanisms of alphavbeta3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human alphavbeta3 (r-alphavbeta3) and compared the activation state of these with alphavbeta3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-alphavbeta3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alphavbeta3 and the receptor in situ on the cell surface. r-alphavbeta3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-alphavbeta3. r-alphavbeta3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native alphavbeta3. On M21-L4 melanoma cells, alphavbeta3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated alphaIIbbeta3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified alphavbeta3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of alphaIIbbeta3 in situ, intracellular controls lower the affinity of alphavbeta3, and the cytoplasmic domains may act as a target for negative regulators of alphavbeta3 activity. PMID:9480902

  3. Ezrin/radixin/moesin proteins are high affinity targets for ADP-ribosylation by Pseudomonas aeruginosa ExoS.

    PubMed

    Maresso, Anthony W; Baldwin, Michael R; Barbieri, Joseph T

    2004-09-10

    Pseudomonas aeruginosa ExoS is a bifunctional type III-secreted cytotoxin. The N terminus (amino acids 96-233) encodes a GTPase-activating protein activity, whereas the C terminus (amino acids 234-453) encodes a factor-activating ExoS-dependent ADP-ribosyltransferase activity. The GTPase-activating protein activity inactivates the Rho GTPases Rho, Rac, and Cdc42 in cultured cells and in vitro, whereas the ADP-ribosylation by ExoS is poly-substrate-specific and includes Ras as an early target for ADP-ribosylation. Infection of HeLa cells with P. aeruginosa producing a GTPase-activating protein-deficient form of ExoS rounded cells, indicating the ADP-ribosyltransferase domain alone is sufficient to elicit cytoskeletal changes. Examination of substrates modified by type III-delivered ExoS identified a 70-kDa protein as an early and predominant target for ADP-ribosylation. Matrix-assisted laser desorption ionization mass spectroscopy identified this protein as moesin, a member of the ezrin/radixin/moesin (ERM) family of proteins. ExoS ADP-ribosylated recombinant moesin at a linear velocity that was 5-fold faster and with a K(m) that was 2 orders of magnitude lower than Ras. Moesin homologs ezrin and radixin were also ADP-ribosylated, indicating the ERMs collectively represent high affinity targets of ExoS. Type III delivered ExoS ADP-ribosylated moesin and ezrin (and/or radixin) in cultured HeLa cells. The ERM proteins contribute to cytoskeleton dynamics, and the ability of ExoS to ADP-ribosylate the ERM proteins links ADP-ribosylation with the cytoskeletal changes associated with ExoS intoxication.

  4. Postthymic expansion in human CD4 naive T cells defined by expression of functional high-affinity IL-2 receptors.

    PubMed

    Pekalski, Marcin L; Ferreira, Ricardo C; Coulson, Richard M R; Cutler, Antony J; Guo, Hui; Smyth, Deborah J; Downes, Kate; Dendrou, Calliope A; Castro Dopico, Xaquin; Esposito, Laura; Coleman, Gillian; Stevens, Helen E; Nutland, Sarah; Walker, Neil M; Guy, Catherine; Dunger, David B; Wallace, Chris; Tree, Timothy I M; Todd, John A; Wicker, Linda S

    2013-03-15

    As the thymus involutes with age, the maintenance of peripheral naive T cells in humans becomes strongly dependent on peripheral cell division. However, mechanisms that orchestrate homeostatic division remain unclear. In this study we present evidence that the frequency of naive CD4 T cells that express CD25 (IL-2 receptor α-chain) increases with age on subsets of both CD31(+) and CD31(-) naive CD4 T cells. Analyses of TCR excision circles from sorted subsets indicate that CD25(+) naive CD4 T cells have undergone more rounds of homeostatic proliferation than their CD25(-) counterparts in both the CD31(+) and CD31(-) subsets, indicating that CD25 is a marker of naive CD4 T cells that have preferentially responded to survival signals from self-Ags or cytokines. CD25 expression on CD25(-) naive CD4 T cells can be induced by IL-7 in vitro in the absence of TCR activation. Although CD25(+) naive T cells respond to lower concentrations of IL-2 as compared with their CD25(-) counterparts, IL-2 responsiveness is further increased in CD31(-) naive T cells by their expression of the signaling IL-2 receptor β-chain CD122, forming with common γ-chain functional high-affinity IL-2 receptors. CD25 plays a role during activation: CD25(+) naive T cells stimulated in an APC-dependent manner were shown to produce increased levels of IL-2 as compared with their CD25(-) counterparts. This study establishes CD25(+) naive CD4 T cells, which are further delineated by CD31 expression, as a major functionally distinct immune cell subset in humans that warrants further characterization in health and disease.

  5. Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human β3 Homomeric GABAA Receptors

    PubMed Central

    Eaton, Megan M.; Cao, Lily Q.; Chen, Ziwei; Franks, Nicholas P.; Evers, Alex S.

    2015-01-01

    Propofol is a sedative and anesthetic agent that can both activate GABAA receptors and potentiate receptor activation elicited by submaximal concentrations of the transmitter. A recent modeling study of the β3 homomeric GABAA receptor postulated a high-affinity propofol binding site in a hydrophobic pocket in the middle of a triangular cleft lined by the M1 and M2 membrane-spanning domains of one subunit and the M2 domain of the neighboring subunit. The goal of the present study was to gain functional evidence for the involvement of this pocket in the actions of propofol. Human β3 and α1β3 receptors were expressed in Xenopus oocytes, and the effects of substitutions of selected residues were probed on channel activation by propofol and pentobarbital. The data demonstrate the vital role of the β3(Y143), β3(F221), β3(Q224), and β3(T266) residues in the actions of propofol but not pentobarbital in β3 receptors. The effects of β3(Y143W) and β3(Q224W) on activation by propofol are likely steric because propofol analogs with less bulky ortho substituents activated both wild-type and mutant receptors. The T266W mutation removed activation by propofol in β3 homomeric receptors; however, this mutation alone or in combination with a homologous mutation (I271W) in the α1 subunit had almost no effect on activation properties in α1β3 heteromeric receptors. We hypothesize that heteromeric α1β3 receptors can be activated by propofol interactions with β3–β3, α1–β3, and β3–α1 interfaces, but the exact locations of the binding site and/or nature of interactions vary in different classes of interfaces. PMID:26206487

  6. High-affinity Anticalins with aggregation-blocking activity directed against the Alzheimer β-amyloid peptide

    PubMed Central

    Rauth, Sabine; Hinz, Dominik; Börger, Michael; Uhrig, Markus; Mayhaus, Manuel; Riemenschneider, Matthias; Skerra, Arne

    2016-01-01

    Amyloid beta (Aβ) peptides, in particular Aβ42 and Aβ40, exert neurotoxic effects and their overproduction leads to amyloid deposits in the brain, thus constituting an important biomolecular target for treatments of Alzheimer's disease (AD). We describe the engineering of cognate Anticalins as a novel type of neutralizing protein reagent based on the human lipocalin scaffold. Phage display selection from a genetic random library comprising variants of the human lipocalin 2 (Lcn2) with mutations targeted at 20 exposed amino acid positions in the four loops that form the natural binding site was performed using both recombinant and synthetic target peptides and resulted in three different Anticalins. Biochemical characterization of the purified proteins produced by periplasmic secretion in Escherichia coli revealed high folding stability in a monomeric state, with Tm values ranging from 53.4°C to 74.5°C, as well as high affinities for Aβ40, between 95 pM and 563 pM, as measured by real-time surface plasmon resonance analysis. The central linear VFFAED epitope within the Aβ sequence was mapped using a synthetic peptide array on membranes and was shared by all three Anticalins, despite up to 13 mutual amino acid differences in their binding sites. All Anticalins had the ability–with varying extent–to inhibit Aβ aggregation in vitro according to the thioflavin-T fluorescence assay and, furthermore, they abolished Aβ42-mediated toxicity in neuronal cell culture. Thus, these Anticalins provide not only useful protein reagents to study the molecular pathology of AD but they also show potential as alternative drug candidates compared with antibodies. PMID:27029347

  7. Synthetic 1,2,3-triazole-linked glycoconjugates bind with high affinity to human galectin-3.

    PubMed

    Marchiori, Marcelo Fiori; Souto, Dênio Emanuel Pires; Bortot, Leandro Oliveira; Pereira, João Francisco; Kubota, Lauro Tatsuo; Cummings, Richard D; Dias-Baruffi, Marcelo; Carvalho, Ivone; Campo, Vanessa Leiria

    2015-07-01

    This work describes the synthesis of the 1,2,3-triazole amino acid-derived-3-O-galactosides 1-6 and the 1,2,3-triazole di-lactose-derived glycoconjugate 7 as potential galectin-3 inhibitors. The target compounds were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-derived amino acids N3-ThrOBn, N3-PheOBn, N3-N-Boc-TrpOBn, N3-N-Boc-LysOBn, N3-O-tBu-AspOBn and N3-l-TyrOH, and the corresponding alkyne-based sugar 3-O-propynyl-GalOMe, as well as by click chemistry reaction between the azido-lactose and 2-propynyl lactose. Surface plasmon resonance (SPR) assays showed that all synthetic glycoconjugates 1-7 bound to galectin-3 with high affinity, but the highest binders were the amino acids-derived glycoconjugates 2 (KD 7.96μM) and 4 (KD 4.56μM), and the divalent lactoside 7 (KD1 0.15μM/KD2 19μM). Molecular modeling results were in agreement with SPR assays, since more stable interactions with galectin-3 were identified for glycoconjugates 2, 4 and 7. Regarding compounds 2 and 4, they established specific cation-π (Arg144) and ionic (Asp148) interactions, whereas glycoconjugate 7 was capable to bridge two independent galectin-3 CRDs, creating a non-covalent cross-link between two monomers and, thus, reaching a submicromolar affinity towards galectin-3.