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Sample records for high-affinity calcium uptake

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil.

    Science.gov (United States)

    Tomatsu, Hajime; Takano, Junpei; Takahashi, Hideki; Watanabe-Takahashi, Akiko; Shibagaki, Nakako; Fujiwara, Toru

    2007-11-20

    Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the K(m) value of MOT1 for molybdate is approximately 20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil.

  4. Effects of anticonvulsants in vivo on high affinity choline uptake in vitro in mouse hippocampal synaptosomes.

    Science.gov (United States)

    Miller, J. A.; Richter, J. A.

    1985-01-01

    The effects of several anticonvulsant drugs on sodium-dependent high affinity choline uptake (HACU) in mouse hippocampal synaptosomes was investigated. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by drugs which have in common the ability to facilitate gamma-aminobutyric acid (GABA) transmission, pentobarbitone, phenobarbitone, barbitone, diazepam, chloridiazepoxide, and valproic acid. Dose-response relationships were determined for these drugs and the drugs' potencies at inhibiting HACU correlated well with their anticonvulsant potencies. Clonazepam, ethosuximide, carbamazepine, and barbituric acid had no effect on HACU in the doses used while phenytoin and trimethadione stimulated HACU. These results suggest that certain anticonvulsants may elicit a part of their anticonvulsant activity by modulating cholinergic neurones. This effect may be mediated through a GABA mechanism. PMID:3978310

  5. The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Casas, C; Aldea, M; Espinet, C; Gallego, C; Gil, R; Herrero, E

    1997-06-15

    High-affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases. Ferrous ions are then transported across the plasma membrane through the FET3 oxidase-FTR1 permease complex. Expression of the high-affinity iron uptake genes is induced upon iron deprivation. We demonstrate that AFT1 is differentially involved in such regulation. Aft1 protein is required for maintaining detectable non-induced level of FET3 expression and for induction of FRE2 in iron starvation conditions. On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi-Iwai et al., EMBO J. 14: 1231-1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way. The alterations in the basal levels of expression of the high-affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources. Overexpression of AFT1 leads to growth arrest of the G1 stage of the cell cycle. Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high-affinity iron uptake genes. Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirement for cell growth.

  6. Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells

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    Mirick Gary

    2009-04-01

    Full Text Available Abstract Background A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL (DvLPBaPPP2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma. Results An analog of the SHAL (DvLPBaPPP2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus. Conclusion The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a

  7. Mitochondrial calcium uptake.

    Science.gov (United States)

    Williams, George S B; Boyman, Liron; Chikando, Aristide C; Khairallah, Ramzi J; Lederer, W J

    2013-06-25

    Calcium (Ca(2+)) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca(2+) levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca(2+) ([Ca(2+)]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca(2+) uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca(2+) uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca(2+) uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca(2+) influx into the mitochondria via the MCU is small relative to other cytosolic Ca(2+) extrusion pathways; (ii) single MCU conductance is ∼6-7 pS (105 mM [Ca(2+)]), and MCU flux appears to be modulated by [Ca(2+)]i, suggesting Ca(2+) regulation of MCU open probability (P(O)); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca(2+) critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca(2+) under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca(2+)]i, mitochondrial Ca(2+) uptake can increase 10- to 1,000-fold and begin to shape [Ca(2+)]i dynamics.

  8. An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil

    OpenAIRE

    Tomatsu, Hajime; Takano, Junpei; Takahashi, Hideki; Watanabe-Takahashi, Akiko; Shibagaki, Nakako; Fujiwara, Toru

    2007-01-01

    Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kine...

  9. CaRuby-Nano: a novel high affinity calcium probe for dual color imaging.

    Science.gov (United States)

    Collot, Mayeul; Wilms, Christian D; Bentkhayet, Asma; Marcaggi, Païkan; Couchman, Kiri; Charpak, Serge; Dieudonné, Stéphane; Häusser, Michael; Feltz, Anne; Mallet, Jean-Maurice

    2015-03-31

    The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

  10. Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

    DEFF Research Database (Denmark)

    Jørgensen, Thomas R; vanKuyk, Patricia A; Poulsen, Bjarne R

    2007-01-01

    This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K(s)) of a reference strain was about 15 microM in glucose-limited chemostat culture. Disruption of mst......-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays...

  11. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    Energy Technology Data Exchange (ETDEWEB)

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.

    1987-12-08

    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity /sup 3/H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light /sup 3/H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib.

  12. The Human Carnitine Transporter SLC22A16 Mediates High Affinity Uptake of the Anticancer Polyamine Analogue Bleomycin-A5*

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    Aouida, Mustapha; Poulin, Richard; Ramotar, Dindial

    2010-01-01

    Bleomycin is used in combination with other antineoplastic agents to effectively treat lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck. However, resistance to bleomycin remains a persistent limitation in exploiting the full therapeutic benefit of the drug with other types of cancers. Previously, we documented that the Saccharomyces cerevisiae l-carnitine transporter Agp2 is responsible for the high affinity uptake of polyamines and of the polyamine analogue bleomycin-A5. Herein, we document that the human l-carnitine transporter hCT2 encoded by the SLC22A16 gene is involved in bleomycin-A5 uptake, as well as polyamines. We show that NT2/D1 human testicular cancer cells, which highly express hCT2, are extremely sensitive to bleomycin-A5, whereas HCT116 human colon carcinoma cells devoid of detectable hCT2 expression or MCF-7 human breast cancer cells that only weakly express the permease showed striking resistance to the drug. NT2/D1 cells accumulated fluorescein-labeled bleomycin-A5 to substantially higher levels than HCT116 cells. Moreover, l-carnitine protected NT2/D1 cells from the lethal effects of bleomycin-A5 by preventing its influx, and siRNA targeted to hCT2 induced resistance to bleomycin-A5-dependent genotoxicity. Furthermore, hCT2 overexpression induced by transient transfection of a functional hCT2-GFP fusion protein sensitized HCT116 cells to bleomycin-A5. Collectively, our data strongly suggest that hCT2 can mediate bleomycin-A5 and polyamine uptake, and that the rate of bleomycin-A5 accumulation may account for the differential response to the drug in patients. PMID:20037140

  13. The human carnitine transporter SLC22A16 mediates high affinity uptake of the anticancer polyamine analogue bleomycin-A5.

    Science.gov (United States)

    Aouida, Mustapha; Poulin, Richard; Ramotar, Dindial

    2010-02-26

    Bleomycin is used in combination with other antineoplastic agents to effectively treat lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck. However, resistance to bleomycin remains a persistent limitation in exploiting the full therapeutic benefit of the drug with other types of cancers. Previously, we documented that the Saccharomyces cerevisiae L-carnitine transporter Agp2 is responsible for the high affinity uptake of polyamines and of the polyamine analogue bleomycin-A5. Herein, we document that the human L-carnitine transporter hCT2 encoded by the SLC22A16 gene is involved in bleomycin-A5 uptake, as well as polyamines. We show that NT2/D1 human testicular cancer cells, which highly express hCT2, are extremely sensitive to bleomycin-A5, whereas HCT116 human colon carcinoma cells devoid of detectable hCT2 expression or MCF-7 human breast cancer cells that only weakly express the permease showed striking resistance to the drug. NT2/D1 cells accumulated fluorescein-labeled bleomycin-A5 to substantially higher levels than HCT116 cells. Moreover, L-carnitine protected NT2/D1 cells from the lethal effects of bleomycin-A5 by preventing its influx, and siRNA targeted to hCT2 induced resistance to bleomycin-A5-dependent genotoxicity. Furthermore, hCT2 overexpression induced by transient transfection of a functional hCT2-GFP fusion protein sensitized HCT116 cells to bleomycin-A5. Collectively, our data strongly suggest that hCT2 can mediate bleomycin-A5 and polyamine uptake, and that the rate of bleomycin-A5 accumulation may account for the differential response to the drug in patients.

  14. Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity zinc uptake system.

    Directory of Open Access Journals (Sweden)

    Cheryl M Vahling-Armstrong

    Full Text Available The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria.

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

    Science.gov (United States)

    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.

  16. ERp57 modulates mitochondrial calcium uptake through the MCU.

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    He, Jingquan; Shi, Weikang; Guo, Yu; Chai, Zhen

    2014-06-01

    ERp57 participates in the regulation of calcium homeostasis. Although ERp57 modulates calcium flux across the plasma membrane and the endoplasmic reticulum membrane, its functions on mitochondria are largely unknown. Here, we found that ERp57 can regulate the expression of the mitochondrial calcium uniporter (MCU) and modulate mitochondrial calcium uptake. In ERp57-silenced HeLa cells, MCU was downregulated, and the mitochondrial calcium uptake was inhibited, consistent with the effect of MCU knockdown. When MCU was re-expressed in the ERp57 knockdown cells, mitochondrial calcium uptake was restored. Thus, ERp57 is a potent regulator of mitochondrial calcium homeostasis.

  17. Mitochondrial calcium uptake capacity modulates neocortical excitability.

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    Sanganahalli, Basavaraju G; Herman, Peter; Hyder, Fahmeed; Kannurpatti, Sridhar S

    2013-07-01

    Local calcium (Ca(2+)) changes regulate central nervous system metabolism and communication integrated by subcellular processes including mitochondrial Ca(2+) uptake. Mitochondria take up Ca(2+) through the calcium uniporter (mCU) aided by cytoplasmic microdomains of high Ca(2+). Known only in vitro, the in vivo impact of mCU activity may reveal Ca(2+)-mediated roles of mitochondria in brain signaling and metabolism. From in vitro studies of mitochondrial Ca(2+) sequestration and cycling in various cell types of the central nervous system, we evaluated ranges of spontaneous and activity-induced Ca(2+) distributions in multiple subcellular compartments in vivo. We hypothesized that inhibiting (or enhancing) mCU activity would attenuate (or augment) cortical neuronal activity as well as activity-induced hemodynamic responses in an overall cytoplasmic and mitochondrial Ca(2+)-dependent manner. Spontaneous and sensory-evoked cortical activities were measured by extracellular electrophysiology complemented with dynamic mapping of blood oxygen level dependence and cerebral blood flow. Calcium uniporter activity was inhibited and enhanced pharmacologically, and its impact on the multimodal measures were analyzed in an integrated manner. Ru360, an mCU inhibitor, reduced all stimulus-evoked responses, whereas Kaempferol, an mCU enhancer, augmented all evoked responses. Collectively, the results confirm aforementioned hypotheses and support the Ca(2+) uptake-mediated integrative role of in vivo mitochondria on neocortical activity.

  18. The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Jacobsen, Christian; Strong, Roland K

    2005-01-01

    in delivering iron to cells during formation of the tubular epithelial cells of the primordial kidney. No cellular receptor for NGAL has been described. We show here that megalin, a member of the low-density lipoprotein receptor family expressed in polarized epithelia, binds NGAL with high affinity, as shown...

  19. Soil carbon content and relative abundance of high affinity H2-oxidizing bacteria predict atmospheric H2 soil uptake activity better than soil microbial community composition

    NARCIS (Netherlands)

    Khdhiri, Mondher; Hesse, Laura; Popa, Maria Elena; Quiza, Liliana; Lalonde, Isabelle; Meredith, Laura K.; Röckmann, Thomas; Constant, Philippe

    2015-01-01

    Soil-atmosphere exchange of H2 is controlled by gas diffusion and the microbial production and oxidation activities in soil. Among these parameters, the H2 oxidation activity catalyzed by soil microorganisms harboring high affinity hydrogenase is the most difficult variable to parameterize because i

  20. Centrifuge-induced hypergravity: [ 3H]GABA and L-[ 14C]glutamate uptake, exocytosis and efflux mediated by high-affinity, sodium-dependent transporters

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    Borisova, T. A.; Himmelreich, N. H.

    The effects of centrifuge-induced hypergravity on the presynaptic events have been investigated in order to provide further insight into regulation of glutamate and GABA neurotransmission and correlation between excitatory and inhibitory responses under artificial gravity conditions. Exposure of animals to hypergravity (centrifugation of rats at 10 G for 1 h) has been found to cause changes in the synaptic processes of brain, in particular neurotransmitter release and uptake in rat brain synaptosomes. Hypergravity loading resulted in more than two-fold enhancement of GABA transporter activity ( Vmax increased from 1.4 ± 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for the animals exposed to hypergravity ( P ⩽ 0.05)). The maximal velocity of L-[ 14C]glutamate uptake decreased from 12.5 ± 3.2 to 5.6 ± 0.9 nmol/min/mg of protein under artificial gravity conditions. Depolarization-evoked exocytotic release of the neurotransmitters has also changed in response to hypergravity. It increased for GABA (7.2 ± 0.54% and 11.74 ± 1.2% of total accumulated label for control and hypergravity, respectively ( P ⩽ 0.05)), but reduced for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%, for control and hypergravity, respectively). Thus, comparative analysis of the neurotransmitter uptake and release has demonstrated that short-term centrifuge-induced 10 G hypergravity loading intensified inhibitory and attenuated excitatory processes in nerve terminals. The activation or reduction of neurotransmitter uptake appeared to be coupled with similarly directed alterations of the neurotransmitter release.

  1. Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation.

    Science.gov (United States)

    Milner, Matthew J; Craft, Eric; Yamaji, Naoki; Koyama, Emi; Ma, Jian Feng; Kochian, Leon V

    2012-07-01

    • In this paper, we conducted a detailed analysis of the ZIP family transporter, NcZNT1, in the zinc (Zn)/cadmium (Cd) hyperaccumulating plant species, Noccaea caerulescens, formerly known as Thlaspi caerulescens. NcZNT1 was previously suggested to be the primary root Zn/Cd uptake transporter. Both a characterization of NcZNT1 transport function in planta and in heterologous systems, and an analysis of NcZNT1 gene expression and NcZNT1 protein localization were carried out. • We show that NcZNT1 is not only expressed in the root epidermis, but also is highly expressed in the root and shoot vasculature, suggesting a role in long-distance metal transport. Also, NcZNT1 was found to be a plasma membrane transporter that mediates Zn but not Cd, iron (Fe), manganese (Mn) or copper (Cu) uptake into plant cells. • Two novel regions of the NcZNT1 promoter were identified which may be involved in both the hyperexpression of NcZNT1 and its ability to be regulated by plant Zn status. • In conclusion, we demonstrate here that NcZNT1 plays a role in Zn and not Cd uptake from the soil, and based on its strong expression in the root and shoot vasculature, could be involved in long-distance transport of Zn from the root to the shoot via the xylem. No claim to original US government works. New Phytologist © 2012 New Phytologist Trust.

  2. High-affinity uptake of kynurenine and nitric oxide-mediated inhibition of indoleamine 2,3-dioxygenase in bone marrow-derived myeloid dendritic cells.

    Science.gov (United States)

    Hara, Toshiaki; Ogasawara, Nanako; Akimoto, Hidetoshi; Takikawa, Osamu; Hiramatsu, Rie; Kawabe, Tsutomu; Isobe, Ken-Ichi; Nagase, Fumihiko

    2008-02-15

    Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway in some dendritic cells (DC) such as plasmacytoid DC (pDC) regulates T-cell responses. It is unclear whether bone marrow-derived myeloid DC (BMDC) express functional IDO. The IDO expression was examined in CD11c(+)CD11b(+) BMDC differentiated from mouse bone marrow cells using GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with the production of nitric oxide (NO) in BMDC in cultures for 24h. In the enzyme assay using cellular extracts of BMDC, the IDO activity of BMDC stimulated with CpG was enhanced by the addition of a NO synthase (NOS) inhibitor, suggesting that IDO activity was suppressed by NO production. On the other hand, the concentration of Kyn in the culture supernatant of BMDC was not increased by stimulation with CpG. Exogenously added Kyn was taken up by BMDC independently of CpG stimulation and NO production, and the uptake of Kyn was inhibited by a transport system L-specific inhibitor or high concentrations of tryptophan. The uptake of tryptophan by BMDC was markedly lower than that of Kyn. In conclusion, IDO activity in BMDC is down-regulated by NO production, whereas BMDC strongly take up exogenous Kyn.

  3. Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1).

    Science.gov (United States)

    Wanichawan, Pimthanya; Hodne, Kjetil; Hafver, Tandekile Lubelwana; Lunde, Marianne; Martinsen, Marita; Louch, William Edward; Sejersted, Ole Mathias; Carlson, Cathrine Rein

    2016-08-01

    NCX1 (Na(+)/Ca(2+) exchanger 1) is an important regulator of intracellular Ca(2+) and a potential therapeutic target for brain ischaemia and for diastolic heart failure with preserved ejection fraction. PLM (phospholemman), a substrate for protein kinases A and C, has been suggested to regulate NCX1 activity. However, although several studies have demonstrated that binding of phosphorylated PLM (pSer(68)-PLM) leads to NCX1 inhibition, other studies have failed to demonstrate a functional interaction of these proteins. In the present study, we aimed to analyse the biological function of the pSer(68)-PLM-NCX1 interaction by developing high-affinity blocking peptides. PLM was observed to co-fractionate and co-immunoprecipitate with NCX1 in rat left ventricle, and in co-transfected HEK (human embryonic kidney)-293 cells. For the first time, the NCX1-PLM interaction was also demonstrated in the brain. PLM binding sites on NCX1 were mapped to two regions by peptide array assays, containing the previously reported PASKT and QKHPD motifs. Conversely, the two NCX1 regions bound identical sequences in the cytoplasmic domain of PLM, suggesting that NCX1-PASKT and NCX1-QKHPD might bind to each PLM monomer. Using two-dimensional peptide arrays of the native NCX1 sequence KHPDKEIEQLIELANYQVLS revealed that double substitution of tyrosine for positions 1 and 4 (K1Y and D4Y) enhanced pSer(68)-PLM binding 8-fold. The optimized peptide blocked binding of NCX1-PASKT and NCX1-QKHPD to PLM and reversed PLM(S68D) inhibition of NCX1 activity (both forward and reverse mode) in HEK-293 cells. Altogether our data indicate that PLM interacts directly with NCX1 and inhibits NCX1 activity when phosphorylated at Ser(68).

  4. Calcium dependent magnesium uptake in myocardium.

    Science.gov (United States)

    Bianchi, C P; Liu, D

    1993-01-01

    The frog myocardium maintains magnesium content at a steady state level when stimulated at 0.4Hz while being perfused with Ringer's solution containing 1 x 10(-3) M Ca2+ and 5 x 10(-7) M magnesium. When calcium is removed 43% of tissue magnesium is lost within 30 seconds or 12 beats. Restoration of calcium to the perfusion solution causes reaccumulation of magnesium from a solution containing 5 x 10(-7) M magnesium. The reaccumulation of magnesium indicates a highly selective transport system for magnesium which is dependent upon the presence of calcium. Calcium appears to reduce the leak of magnesium from the myocardium and enhances the transport of magnesium into the myocardial cell. Intracellular magnesium is a necessary cofactor for hundreds of enzymes, and is essential for protein synthesis and as an extracellular divalent cation helps to stabilize excitable membranes in conjunction with calcium. The concentration of ionized magnesium in the sarcoplasm of myocardial muscle has an average value of 1.45 mM +/- 1.37 (standard deviation), N = 19) with a range of 0.5 to 3.6 mM (1). The heart with its numerous mitochondria and high enzymatic activity is vulnerable to myocardial damage due to magnesium loss. The isolated frog ventricle conserves intracellular magnesium when perfused with Ringer's solution containing no added magnesium and maintains function for hours. The ability to conserve magnesium suggests a low permeability of the sarcolemma to magnesium and an extremely efficient inward transport system. Removal of calcium as well as magnesium from the perfusion solution causes a rapid loss of tension in the electrically driven frog ventricle (0.4) Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Silver Nanoparticle-Directed Mast Cell Degranulation Is Mediated through Calcium and PI3K Signaling Independent of the High Affinity IgE Receptor.

    Science.gov (United States)

    Alsaleh, Nasser B; Persaud, Indushekhar; Brown, Jared M

    2016-01-01

    Engineered nanomaterial (ENM)-mediated toxicity often involves triggering immune responses. Mast cells can regulate both innate and adaptive immune responses and are key effectors in allergic diseases and inflammation. Silver nanoparticles (AgNPs) are one of the most prevalent nanomaterials used in consumer products due to their antimicrobial properties. We have previously shown that AgNPs induce mast cell degranulation that was dependent on nanoparticle physicochemical properties. Furthermore, we identified a role for scavenger receptor B1 (SR-B1) in AgNP-mediated mast cell degranulation. However, it is completely unknown how SR-B1 mediates mast cell degranulation and the intracellular signaling pathways involved. In the current study, we hypothesized that SR-B1 interaction with AgNPs directs mast cell degranulation through activation of signal transduction pathways that culminate in an increase in intracellular calcium signal leading to mast cell degranulation. For these studies, we utilized bone marrow-derived mast cells (BMMC) isolated from C57Bl/6 mice and RBL-2H3 cells (rat basophilic leukemia cell line). Our data support our hypothesis and show that AgNP-directed mast cell degranulation involves activation of PI3K, PLCγ and an increase in intracellular calcium levels. Moreover, we found that influx of extracellular calcium is required for the cells to degranulate in response to AgNP exposure and is mediated at least partially via the CRAC channels. Taken together, our results provide new insights into AgNP-induced mast cell activation that are key for designing novel ENMs that are devoid of immune system activation.

  6. Calcium Uptake in Crude Tissue Preparation

    Science.gov (United States)

    Bidwell, Philip A.; Kranias, Evangelia G.

    2016-01-01

    SUMMARY The various isoforms of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) are responsible for the Ca2+ uptake from the cytosol into the endoplasmic or sarcoplasmic reticulum (ER/SR). In some tissues, the activity of SERCA can be modulated by binding partners, such as phospholamban and sarcolipin. The activity of SERCA can be characterized by its apparent affinity for Ca2+ as well as maximal enzymatic velocity. Both parameters can be effectively determined by the protocol described here. Specifically, we describe the measurement of the rate of oxalate-facilitated 45Ca uptake into the SR of crude mouse ventricular homogenates. This protocol can easily be adapted for different tissues and animal models as well as cultured cells. PMID:26695031

  7. Regulation of nutrient uptake, water uptake and growth under calcium starvation and recovery

    NARCIS (Netherlands)

    Amor, del F.M.; Marcelis, L.F.M.

    2003-01-01

    To analyze the dynamics of growth, water and nutrient uptake, the effects of 1, 3 and 7 d of calcium starvation and the recovery capability during 7 d afterwards were investigated in vegetative tomato plants. Results showed that after only 1 d of Ca-starvation, leaf photosynthesis, leaf expansion an

  8. Calcium uptake in brain synaptosomes: a pharmacologic study

    Energy Technology Data Exchange (ETDEWEB)

    Rampe, D.E.

    1986-01-01

    Pinched-off nerve endings (synaptosomes) from rat and guinea pig brain were used as a model to study Ca/sup 2 +/ entry mechanisms in neuronal tissue. Synaptosomes contain high affinity binding sites for both, 1,4-dihydropyridine Ca/sup 2 +/ channel antagonists, and activators. The thermodynamic characteristics of (/sup 3/H)nitrendipine building in synaptosomes were similar to those seen in both cardiac and smooth muscle preparations. Synaptosomes display two distinct K/sup +/-induced Ca/sup 2 +/ entry mechanisms. These are kinetically distinct with the faster of the two terminating in approx. 1 second while the slower persists for approx. minute. The slow phase uptake process is abolished in Na/sup +/-free media, is sensitive to antagonism by 3,4-dichlorobenzamil and displays a more rapid ontogenic appearance relative to the fast phase. It is likely that the slow phase represents Ca/sup 2 +/ entry via Na/sup +//Ca/sup 2 +/ exchange. The rapid inactivation of the fast phase coupled with its voltage dependence suggest that it represents Ca/sup 2 +/ entry via one or more types of voltage dependent Ca/sup 2 +/ channels. These channels may not be dihydropyridin sensitive since neither nitrendipine nor Bay K 8644 were shown to modulate synaptosomal Ca/sup 2 +/ uptake. The benzodiazepine receptor ligands Ro 5-4864, PK 11195 and diazepam all selectively inhibited fast phase Ca/sup 2 +/ entry relative to slow phase entry. In addition, these compounds altered (/sup 3/H)nitrendipine binding affinity. It is concluded that certain benzodiazepine receptor ligands can interact specifically with voltage dependent Ca/sup 2 +/ channels.

  9. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

    Science.gov (United States)

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A S; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-06-09

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels.

  10. Inulin and fructooligosaccharide affect in vitro calcium uptake and absorption from calcium-enriched gluten-free bread.

    Science.gov (United States)

    Krupa-Kozak, U; Swiątecka, D; Bączek, N; Brzóska, M M

    2016-04-01

    Compromised intestinal calcium absorption affecting a deterioration of bone state is a sign of coeliac disease. Experimental calcium-fortified gluten-free bread (GFB) of improved calcium bioavailability could increase calcium content in the diets of coeliac disease patients, allowing them to obtain the amount of calcium they need for therapeutic use. Prebiotics, including inulin-type fructans (IFs) have a beneficial effect on calcium bioavailability. In the present study, the in vitro model composed of the intestinal-like Caco-2 cells and the human intestinal bacteria (Lactobacillus, Enterococcus and Enterobacteriaceae) were used to analyse the effect of inulin and fructooligosaccharide (FOS) of different chain lengths, on calcium uptake and absorption from experimental GFB. Analysed IFs, especially short-chain FOS, significantly (p < 0.05) increased cellular calcium uptake from GFB digest and stimulated the intestinal bacteria applied in the cultures to the intensive synthesis of organic acids. In particular, the concentration of butyric, valeric and lactic acids increased significantly. Similarly, in the calcium absorption experiment, IFs increased the cellular calcium retention but concomitantly reduced its content in basolateral filtrates. The results obtained suggest that the applied IFs affected differentially calcium uptake and absorption from the experimental calcium-enriched GFB, therefore a further study is needed to assess whether these observations made in vitro contribute to IF effects on calcium absorption from experimental GFB in vivo.

  11. Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Peter Rohloff

    Full Text Available Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+/H(+ countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

  12. The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

    Science.gov (United States)

    Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

    Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

  13. Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals.

    Science.gov (United States)

    Marland, Jamie Roslin Keynes; Hasel, Philip; Bonnycastle, Katherine; Cousin, Michael Alan

    2016-01-29

    Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

  14. High affinity complexes of pannexin channels and L-type calcium channel splice-variants in human lung: Possible role in clevidipine-induced dyspnea relief in acute heart failure

    Directory of Open Access Journals (Sweden)

    Gerhard P. Dahl

    2016-08-01

    Research in Context: Clevidipine lowers blood pressure by inhibiting calcium channels in vascular smooth muscle. In patients with acute heart failure, clevidipine was shown to relieve breathing problems. This was only partially related to the blood pressure lowering actions of clevidipine and not conferred by another calcium channel inhibitor. We here found calcium channel variants in human lung that are more selectively inhibited by clevidipine, especially when associated with pannexin channels. This study gives a possible mechanism for clevidipine's relief of breathing problems and supports future clinical trials testing the role of clevidipine in the treatment of acute heart failure.

  15. Desalted duck egg white peptides promote calcium uptake by counteracting the adverse effects of phytic acid.

    Science.gov (United States)

    Hou, Tao; Liu, Weiwei; Shi, Wen; Ma, Zhili; He, Hui

    2017-03-15

    The structure of the desalted duck egg white peptides-calcium chelate was characterized by fluorescence spectroscopy, fourier transform infrared spectroscopy, and dynamic light scattering. Characterization results showed structural folding and aggregation of amino acids or oligopeptides during the chelation process. Desalted duck egg white peptides enhanced the calcium uptake in the presence of oxalate, phosphate and zinc ions in Caco-2 monolayers. Animal model indicated that desalted duck egg white peptides effectively enhanced the mineral absorption and counteracted the deleterious effects of phytic acid. These findings suggested that desalted duck egg white peptides might promote calcium uptake in three pathways: 1) desalted duck egg white peptides bind with calcium to form soluble chelate and avoid precipitate; 2) the chelate is absorbed as small peptides by enterocyte; and 3) desalted duck egg white peptides regulate the proliferation and differentiation of enterocytes through the interaction with transient receptor potential vanilloid 6 calcium channel.

  16. Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake.

    Science.gov (United States)

    Wang, Lele; Yang, Xue; Li, Siwei; Wang, Zheng; Liu, Yu; Feng, Jianrong; Zhu, Yushan; Shen, Yuequan

    2014-03-18

    Mitochondrial calcium uptake is a critical event in various cellular activities. Two recently identified proteins, the mitochondrial Ca(2+) uniporter (MCU), which is the pore-forming subunit of a Ca(2+) channel, and mitochondrial calcium uptake 1 (MICU1), which is the regulator of MCU, are essential in this event. However, the molecular mechanism by which MICU1 regulates MCU remains elusive. In this study, we report the crystal structures of Ca(2+)-free and Ca(2+)-bound human MICU1. Our studies reveal that Ca(2+)-free MICU1 forms a hexamer that binds and inhibits MCU. Upon Ca(2+) binding, MICU1 undergoes large conformational changes, resulting in the formation of multiple oligomers to activate MCU. Furthermore, we demonstrate that the affinity of MICU1 for Ca(2+) is approximately 15-20 μM. Collectively, our results provide valuable details to decipher the molecular mechanism of MICU1 regulation of mitochondrial calcium uptake.

  17. Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels

    OpenAIRE

    2008-01-01

    Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure plan...

  18. Dyes with high affinity for polylactide

    Institute of Scientific and Technical Information of China (English)

    Liang He; Shu Fen Zhang; Bing Tao Tang; Li Li Wang; Jin Zong Yang

    2007-01-01

    Attempts were made to develop dyes with high affinity for polylactide as an alternative to the existent commercial disperse dyes.The dyes synthesized according to the affinity concept of dye to polylactide exhibited excellent dyeing properties on polylactide compared with the commercial disperse dyes.

  19. Effect of glucose stimulation on /sup 45/calcium uptake of rat pancreatic islets and their total calcium content as measured by a fluorometric micro-method

    Energy Technology Data Exchange (ETDEWEB)

    Wolters, G.H.J.; Wiegman, J.B.; Konijnendijk, W.

    1982-02-01

    Glucose-stimulated /sup 45/calcium uptake and total calcium content of rat pancreatic islets has been studied, using a new fluorometric micro-method to estimate total calcium. Extracellular calcium was separated from incubated tissue by a rapid micro-filtration procedure. Islets incubated up to 60 min with calcium chloride 2.5 mmol/l and glucose 2.5 mmol/l maintained the same calcium content (670 +- 7.5 pmol/..mu..g DNA). When the glucose concentration was raised to 15 mmol/l no change in the total calcium content could be detected. On incubation with glucose 2.5 mmol/l in the absence of calcium, the calcium content decreased to 488 +- 27 pmol/..mu..g DNA. On incubation with /sup 45/calcium chloride 2.5 mmol/l for 5 or 30 min at 2.5 mmol/l glucose, islets exchanged 21 +- 2 and 28 +- 1% of their total calcium content and, at 15 mmol/l glucose, 30 +- 3 and 45 +- 2%, respectively. Thus, islet calcium has a high turn-over rate. Glucose stimulation results in an increase of the calcium uptake without enhancing the total calcium content and hence must increase the calcium-exchangeable pool.

  20. The calcium-induced conformation and glycosylation of scavenger-rich cysteine repeat (SRCR) domains of glycoprotein 340 influence the high affinity interaction with antigen I/II homologs.

    Science.gov (United States)

    Purushotham, Sangeetha; Deivanayagam, Champion

    2014-08-01

    Oral streptococci adhere to tooth-immobilized glycoprotein 340 (GP340) via the surface protein antigen I/II (AgI/II) and its homologs as the first step in pathogenesis. Studying this interaction using recombinant proteins, we observed that calcium increases the conformational stability of the scavenger-rich cysteine repeat (SRCRs) domains of GP340. Our results also show that AgI/II adheres specifically with nanomolar affinity to the calcium-induced SRCR conformation in an immobilized state and not in solution. This interaction is significantly dependent on the O-linked carbohydrates present on the SRCRs. This study also establishes that a single SRCR domain of GP340 contains the two surfaces to which the apical and C-terminal regions of AgI/II noncompetitively adhere. Compared with the single SRCR domain, the three tandem SRCR domains displayed a collective/cooperative increase in their bacterial adherence and aggregation. The previously described SRCRP2 peptide that was shown to aggregate several oral streptococci displayed limited aggregation and also nonspecific adherence compared to SRCR domains. Finally, we show distinct species-specific adherence/aggregation between Streptococcus mutans AgI/II and Streptococcus gordonii SspB in their interaction with the SRCRs. This study concludes that identification of the metal ion and carbohydrate adherence motifs on both SRCRs and AgI/II homologs could lead to the development of anti-adhesive inhibitors that could deter the adherence of pathogenic oral streptococci and thereby prevent the onset of infections.

  1. Organic acid effect on calcium uptake by the wheat roots

    Directory of Open Access Journals (Sweden)

    Fabrize Caroline Nunes

    2009-02-01

    Full Text Available This work evaluated the effect of the natural organic acids on the uptake of Ca by the wheat roots in a hydroponic solution. The following organic acids were evaluated: citric, oxalic, tartaric, malic, malonic, maleic, DL-malic, p-hydroxybenzoic, aconitic, and salicilic. The organic acids neither enhanced the root growth nor increased Ca uptake. The salicilic and malic acids were highly toxic and decreased the root growth. The citric, tartaric, maleic, aconitic, and salicilic decreased the Ca uptake by the roots due to their higher capacity to form the stable complexes with Ca in solution at pH 6.0. Decreasing the Ca valence from Ca++ to CaL+ or CaL2(0 through the organic ligand complexation reactions decreased the Ca uptake. The results suggested that the wheat roots do not absorb Ca-organic complexes.Ácidos orgânicos possuem grupos funcionais com cargas negativas que complexam íons metálicos em solução. Este trabalho avaliou o efeito de ácidos orgânicos naturais na absorção de Ca pelas raízes de trigo. Foram avaliados os seguintes ácidos orgânicos: cítrico, oxálico, tartarico, málico, malônico, maleico, DL-málico, p-hidroxibenzoico, aconítico e salicílico. Os ácidos orgânicos não estimularam o crescimento das raízes e não aumentaram a absorção de Ca. Os ácidos salicílico e maleico diminuíram drasticamente o crescimento radicular. Os ácidos cítrico, tartárico, maleico, aconítico e salicílico diminuíram a absorção de Ca pelas raízes devido à maior capacidade de formar complexos estáveis com Ca em solução no pH 6,0. A redução da valência de Ca++ para CaL+ e CaL2(0, através das reações de complexação, diminuiu a absorção de Ca pelas raízes. Os resultados sugerem que os complexos de Ca-orgânico não são absorvidos pelas raízes de trigo.

  2. Hydrogen sulfide promotes calcium uptake in larval zebrafish.

    Science.gov (United States)

    Kwong, Raymond W M; Perry, Steve F

    2015-07-01

    Hydrogen sulfide (H2S) can act as a signaling molecule for various ion channels and/or transporters; however, little is known about its potential involvement in Ca(2+) balance. Using developing zebrafish (Danio rerio) as an in vivo model system, the present study demonstrated that acute exposure to H2S donors increased Ca(2+) influx at 4 days postfertilization, while chronic (3-day) exposure caused a rise in whole body Ca(2+) levels. The mRNA expression of Ca(2+)-transport-related genes was unaffected by H2S exposure, suggesting that posttranscriptional modifications were responsible for the altered rates of Ca(2+) uptake. Indeed, treatment of fish with the protein kinase A inhibitor H-89 abolished the H2S-mediated stimulation of Ca(2+) influx, suggesting that H2S increased Ca(2+) influx by activating cAMP-protein kinase A pathways. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are two key enzymes in the endogenous synthesis of H2S. Using an antisense morpholino knockdown approach, we demonstrated that Ca(2+) influx was reduced in CBS isoform b (CBSb)- but not in CSE-deficient fish. Interestingly, the reduction in Ca(2+) influx in CBSb-deficient fish was observed only in fish that were acclimated to low-Ca(2+) water (i.e., 25 μM Ca(2+); control: 250 μM Ca(2+)). Similarly, mRNA expression of cbsb but not cse was increased in fish acclimated to low-Ca(2+) water. Results from whole-mount immunohistochemistry further revealed that CBSb was expressed in Na(+)-K(+)-ATPase-rich cells, which are implicated in Ca(2+) uptake in zebrafish larvae. Collectively, the present study suggests a novel role for H2S in promoting Ca(2+) influx, particularly in a low-Ca(2+) environment.

  3. Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels.

    Science.gov (United States)

    Arispe, Nelson; Diaz, Juan Carlos; Simakova, Olga; Pollard, Harvey B

    2008-02-19

    Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure planar phospholipid bilayers. These digitoxin channels are blocked by Al(3+) and La(3+) but not by Mg(2+) or the classical l-type calcium channel blocker, nitrendipine. In bilayers, we find that the chemistry of the lipid affects the kinetics of the digitoxin channel activity, but not the cation selectivity. Antibodies against digitoxin promptly neutralize digitoxin channels in both cells and bilayers. We propose that these digitoxin calcium channels may be part of the mechanism by which digitoxin and other active cardiac glycosides, such as digoxin, exert system-wide actions at and above the therapeutic concentration range.

  4. Exploration of As(III)/As(V) Uptake from Aqueous Solution by Synthesized Calcium Sulfate Whisker☆

    Institute of Scientific and Technical Information of China (English)

    Xiaojuan Chen; Liuchun Yang; Junfeng Zhang; Yan Huang

    2014-01-01

    Although common calcium-containing minerals such as calcite and gypsum may fix arsenic, the interaction be-tween modified calcic minerals and arsenic has seldom been reported. The uptake behavior of As(III)/As(V) from aqueous solutions by calcium sulfate whisker (CSW, dihydrate or anhydrite) synthesized through a cooling recrystal ization method was explored. A series of batch experiments were conducted to examine the effect of pH, reaction time, whisker dosage, and initial As concentration. X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used to characterize the samples prepared. The results showed that pH of the aqueous solution was an important parameter for As(III)/As(V) uptake, and an excellent removal efficiency could be achieved under strongly alkaline condition. The data from batch experiments for reaction of As(V) with calcium sulfate dihydrate whisker (CSDW) and calcium sulfate anhydrous whisker (CSAW) were well described with extended Langmuir EXT1 model, from which theoretic maximum adsorption capacity of 46.57 mg As(V)·(g CSDW)−1 and 39.18 mg As(V)·(g CSAW)−1 were obtained. Some calcium arsenate solids products, such as CaAsO3(OH) (weilite, syn), Ca3(AsO4)2 (calcium arsenate), CaO–As2O5, Ca–As–O, Ca5(AsO4)3OH·xH2O (calcium arsenate hydroxide hydrate), and CaH(AsO4)·2H2O (hydrogen calcium arsenic oxide hydrate), were detected at pH = 12.5 through XRD analysis. This indicates that the interaction mechanism between As(V) and CSW is a complex adsorption process combined with surface dissolution and chemical precipitation.

  5. Silver Uptake, Distribution, and Effect on Calcium, Phosphorus, and Sulfur Uptake 1

    Science.gov (United States)

    Koontz, Harold V.; Berle, Karen L.

    1980-01-01

    Bean, corn, and tomato plants were grown in a nutrient solution labeled with 32P, 45Ca, or 35S and varying concentrations of AgNO3. Following a 6-hour treatment period, plants were harvested and analyzed. A low Ag+ concentration (50 nanomolar) inhibited the shoot uptake of the ions investigated. In the roots, Ca uptake increased whereas P and S uptake decreased. Autoradiograms of bean and corn plants, using 110mAg, showed that Ag+ was uniformly deposited in the bean shoot, but corn shoots had regions of high activity along the leaf margins and at the tips where guttation had occurred. Roots were heavily labeled and shoots (especially the new growth) continued to accumulate Ag+ even after the intact plant was returned to Ag-free solution. Silver was believed to be phloem-mobile since it was exported from a treated leaf. Bean plants removed one-half the Ag+ from 4 liters of nutrient solution containing 50 nanomolar AgNO3 within 1.5 hours, but took 16 hours for 20 liters of solution. Images PMID:16661185

  6. Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells.

    Science.gov (United States)

    Khanal, Ramesh C; Peters, Tremaine M Sterling; Smith, Nathan M; Nemere, Ilka

    2008-11-01

    Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.

  7. Effects of antibiotics on uptake of calcium into isolated nerve terminals

    Energy Technology Data Exchange (ETDEWEB)

    Atchison, W.D.; Adgate, L.; Beaman, C.M.

    1988-05-01

    The goal of the present study was to determine whether several antibiotics which are known to block neuromuscular transmission would impair depolarization-dependent and/or -independent uptake of calcium into isolated nerve terminals prepared from forebrain synaptosomes of rats by conventional methods. Antibiotics tested for potential block of Ca++ uptake included the aminoglycosides neomycin and streptomycin, the lincosamide clindamycin, oxytetracycline and polymyxin B. Drugs were applied in concentrations ranging from 1 to 1000 microM. Uptake of 45Ca was determined during depolarization induced by an elevated K+ concentration (77.5 mM). Influxes of 45Ca during 1 and 10 sec of depolarization were used to assess Ca++ uptake via a fast, inactivating path and total uptake, respectively. Uptake of 45Ca during 10 sec of depolarization into synaptosomes which were previously depolarized for 10 sec in the presence of 77.5 mM K+ but in the absence of external Ca++ was used to measure uptake during a slow, noninactivating path. Total depolarization-dependent uptake of 45Ca was depressed significantly by all antibiotics tested except oxytetracycline; however, the various agents differed with respect to their efficacy and potency as blockers of Ca influx. The fast component of uptake, which is thought to be associated with neurotransmitter release, was decreased significantly by all antibiotics. Neomycin and polymyxin were the most potent and most effective at lowering fast phase 45Ca influx; streptomycin, was intermediate in effectiveness whereas clindamycin and oxytetracycline were only effective at concentrations greater than or equal to 100 microM. Only clindamycin, streptomycin and polymyxin B caused significant reductions in the slow phase of 45Ca uptake.

  8. Tree species effects on calcium cycling: The role of calcium uptake in deep soils

    NARCIS (Netherlands)

    Dijkstra, F.A.; Smits, M.M.

    2002-01-01

    Soil acidity and calcium (Ca) availability in the surface soil differ substantially beneath sugar maple (Acer saccharum) and eastern hemlock (Tsuga canadensis) trees in a mixed forest in northwestern Connecticut. We determined the effect of pumping of Ca from deep soil (rooting zone below 20-cm

  9. Calcium uptake by sarcoplasmic reticulum in the presence of organophosphorus insecticide methyl-parathion

    Energy Technology Data Exchange (ETDEWEB)

    Blasiak, J. [Lodz Univ. (Poland)

    1995-12-31

    Using an isotope labelling technique it has been shown that an organophosphorus insecticide methyl parathion (0,0-diethyl 0-4-nitrophenyl phosphorothionate) depressed calcium uptake by sarcoplasmic reticulum isolated from rabbit hind leg muscle. The effect was significant for insecticide concentrations of 50 and 100 {mu}M and was dose-dependent. The insecticide exerted a more pronounced effect on calcium uptake in the presence of ATP in the reticulum environment than in the absence of ATP. The inhibitory action of methyl parathion on Ca{sup 2+} accumulation by sarcoplasmic reticulum can cause a rise in myoplasmic free Ca{sup 2+}, the essential prerequisite for contracture activation. Because methyl parathion, as well as other organophosphorus insecticides, is primarily neurotoxic, evidence of non-specific effect could be important for assessing its environmental safety. (author). 20 refs, 2 figs.

  10. Interaction of lead and calcium uptake by the woodlouse, Porcellio scaber (Isopoda, porcellionidae)

    Energy Technology Data Exchange (ETDEWEB)

    Beeby, A.

    1978-01-01

    Lead and calcium nitrate in a potato base were fed to Porcellio scaber; three levels of each cation were used in a 3/sup 2/ factorial design experiment. The amounts accumulated were determined by atomic absorption spectrophotometric analysis of nitric acid digests of whole animals. Both cations increased markedly in the woodlice with increasng dosage and a clear correlation was found between the rates of uptake of the two.

  11. Uptake of (N-Me-3H)-choline by synaptosomes from the central nervous system of Locusta migratoria

    Energy Technology Data Exchange (ETDEWEB)

    Breer, H.

    1982-03-01

    The accumulation of 3H-choline by isolated synaptosomes from the central nervous system of locust was studied at concentrations varying from 0.05 to 40 microM. Kinetic analysis of the saturable process revealed a high-affinity and a low-affinity system. The high-affinity uptake was competitively inhibited by hemicholinium-3 and was absolutely dependent on external sodium. Elevated potassium concentrations inhibited choline uptake. The choline uptake by insect synaptosomes was found to be remarkably resistant to a variety of metabolic inhibitors. The reduced choline uptake under depolarizing conditions (high potassium concentration or veratridine) in the absence of calcium implies that electrochemical gradients are important for high-affinity choline uptake. Depolarization of preloaded synaptosomes under appropriate conditions resulted in a significant release of newly accumulated choline radioactivity.

  12. Encapsulation of plasmid DNA in calcium phosphate nanoparticles: stem cell uptake and gene transfer efficiency

    Directory of Open Access Journals (Sweden)

    Cao X

    2011-12-01

    Full Text Available Xia Cao*, Wenwen Deng*, Yuan Wei*, Weiyan Su, Yan Yang, Yawei Wei, Jiangnan Yu, Ximing XuDepartment of Pharmaceutics, School of Pharmacy, and Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Jingkou District, Zhenjiang, People's Republic of China*These authors contributed equally to this workBackground: The purpose of this study was to develop calcium phosphate nanocomposite particles encapsulating plasmid DNA (CP-pDNA nanoparticles as a nonviral vector for gene delivery.Methods: CP-pDNA nanoparticles employing plasmid transforming growth factor beta 1 (TGF-β1 were prepared and characterized. The transfection efficiency and cell viability of the CP-pDNA nanoparticles were evaluated in mesenchymal stem cells, which were identified by immunofluorescence staining. Cytotoxicity of plasmid TGF-β1 and calcium phosphate to mesenchymal stem cells were evaluated by MTT assay.Results: The integrity of TGF-β1 encapsulated in the CP-pDNA nanoparticles was maintained. The well dispersed CP-pDNA nanoparticles exhibited an ultralow particle size (20–50 nm and significantly lower cytotoxicity than Lipofectamine™ 2000. Immunofluorescence staining revealed that the cultured cells in this study were probably mesenchymal stem cells. The cellular uptake and transfection efficiency of the CP-pDNA nanoparticles into the mesenchymal stem cells were higher than that of needle-like calcium phosphate nanoparticles and a standard calcium phosphate transfection kit. Furthermore, live cell imaging and confocal laser microscopy vividly showed the transportation process of the CP-pDNA nanoparticles in mesenchymal stem cells. The results of a cytotoxicity assay found that both plasmid TGF-β1 and calcium phosphate were not toxic to mesenchymal stem cells.Conclusion: CP-pDNA nanoparticles can be developed into an effective alternative as a nonviral gene delivery system that is highly efficient and has low cytotoxicity.Keywords: calcium

  13. Characterization of a genetically reconstituted high-affinity system for serotonin transport

    Energy Technology Data Exchange (ETDEWEB)

    Chang, A.S.S.; Lam, D.M.K. (Baylor College of Medicine, Woodlands, TX (USA) Baylor College of Medicine, Houston, TX (USA)); Frnka, J.V.; Chen, D. (Baylor College of Medicine, Woodlands, TX (USA))

    1989-12-01

    By transfecting mouse fibroblast L-M cells with human genomic DNA, the authors have established and identified several clonal cell lines that stably express a high-affinity serotonin (5-HT)-uptake mechanism absent in untransfected host cells. One such cell line, L-S1, possesses features of 5-({sup 3}H)HT uptake similar to those previously characterized in the central nervous system and blood platelets: (i) specificity for 5-HT; (ii) antagonism by imipramine, a known inhibitor of high-affinity 5-HT uptake; (iii) both Na{sup +} and temperature dependence; (iv) kinetic saturability; and (v) high affinity for 5-HT. This cell line can be used to compare the relative efficacies of known blockers of 5-HT uptake and thereby offers a rapid and reliable assay system for testing novel inhibitors of this system. Since L-S1 contains stably integrated human DNA in its genome, they postulate that the observed 5-HT-uptake system resulted from the expression of human gene(s) coding for the 5-HT transporter. Thus, cell lines such as L-S1 may represent novel means for screening and developing therapeutic agents specific for neutrotransmitter-uptake systems as well as substrate for the cloning and elucidation of the genes encoding the various neurotransmitter transporters.

  14. Rapid responses to reverse T₃ hormone in immature rat Sertoli cells: calcium uptake and exocytosis mediated by integrin.

    Directory of Open Access Journals (Sweden)

    Ana Paula Zanatta

    Full Text Available There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T₃ (rT₃ action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT₃ are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT₃ was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT₃-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions. Therefore, our findings suggest that calcium uptake stimulated by rT₃ may be mediated by integrin αvβ₃. In addition, it was demonstrated that calcium uptake stimulated by rT₃ is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT₃ also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT₃ modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.

  15. Calcium acetate induces calcium uptake and formation of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L.

    Science.gov (United States)

    Borchert, R

    1986-09-01

    During treatment of isolated, peeled leaflets of Gleditsia triacanthos with 0.5-2 mM [(45)Ca]acetate, saturation of the cell-wall free space with Ca(2+) occurred within 10 min and was followed by a period of 6-10 h during which there was no significant Ca-uptake into the protoplast, but apoplastic Ca(2+) was periodically released into the medium. Later, Ca(2+) was absorbed for 3-4 d at rates of up to 2.2 μmol Ca(2+)·h(-1)·(g FW)(-1) to final concentrations of 350 μmol Ca(2+)· (g FW)(-1). The distribution of absorbed Ca(2+) between cell wall, vacuole and Ca-oxalate crystals was determined during Ca-uptake. Wheras intact, cut leaflets deposited absorbed Ca(2+) as Ca-oxalate in the crystal cells, peeled leaflets lacking crystal cells accumulated at least 40-50 μmol·(g FW)(-1) soluble Ca(2+) before the absorbed Ca(2+) was precipitated as Ca-oxalate. These observations indicate that the mechanisms for the continuous uptake of Ca(2+), the synthesis of oxalate and the precipitation of Ca(2+) as Ca-oxalate are operational in the crystal cells of intact leaflets, but not in the mesophyll cells of peeled leaflets where they must be induced by exposure to Ca(2+). The precipitation of absorbed Ca(2+) as Ca-oxalate by the crystal cells of isolated Gleditsia leaflets illustrates the role of these cells in the excretion of surplus Ca(2+) which enters normal, attached leaves with the transpiration stream.In addition to acetate, only Ca-lactate and Ca-carbonate lead to Ca-uptake, but at rates well below those observed with Ca-acetate. Other small organic anions (citrate, glycolate, glyoxalate, malate) and inorganic anions (chloride, nitrate, sulfate) did not permit Ca-uptake. Acetate-(14)C was rapidly absorbed during Ca-uptake, but less than 20% was incorporated into Ca-oxalate; the rest remained mostly in the soluble fraction or was metabolized to CO2. Acetate, as a permeable weak acid, may enable rapid Ca-uptake by stimulating proton extrusion at the plasmalemma and by

  16. 3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

    Science.gov (United States)

    Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

    2012-05-01

    3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.

  17. Maternal provision and embryonic uptake of calcium in an oviparous and a placentotrophic viviparous Australian lizard (Lacertilia: Scincidae).

    Science.gov (United States)

    Stewart, James R; Ecay, Tom W; Garland, Courtney P; Fregoso, Santiago P; Price, Elizabeth K; Herbert, Jacquie F; Thompson, Michael B

    2009-06-01

    Embryos of oviparous lizards have two sources of calcium for embryonic development: 1) calcium that accumulates in yolk during vitellogenesis, and 2) calcium carbonate deposited in the eggshell from oviductal secretions. Eggs of viviparous lizards lack a calcified eggshell and calcium secreted by the uterus is delivered to the embryo across a placenta. Whereas oviparous lizard embryos recover calcium from the eggshell during late developmental growth stages, viviparous embryos have a lengthy intimate association with the uterus and the potential for an extended interval of placental calcium transfer. We compared the pattern of calcium mobilization of embryos of the viviparous, placentotrophic scincid lizard, Pseudemoia pagenstecheri, to that of a closely related oviparous species, Saproscincus mustelinus, to determine if the timing of uterine calcium secretion was influenced by reproductive mode. Embryos of both species receive a substantial amount of calcium from either the eggshell or placenta (54% and 85% respectively). The ontogeny of calcium uptake by embryos of P. pagenstecheri reveals that the onset of embryonic acquisition of calcium occurs earlier relative to embryonic stage but the timing of peak uterine secretion of calcium is delayed, compared to S. mustelinus.

  18. Uptake of the proteins HTRA1 and HTRA2 by cells mediated by calcium phosphate nanoparticles

    Science.gov (United States)

    Rotan, Olga; Severin, Katharina N; Pöpsel, Simon; Peetsch, Alexander; Merdanovic, Melisa; Ehrmann, Michael

    2017-01-01

    The efficient intracellular delivery of (bio)molecules into living cells remains a challenge in biomedicine. Many biomolecules and synthetic drugs are not able to cross the cell membrane, which is a problem if an intracellular mode of action is desired, for example, with a nuclear receptor. Calcium phosphate nanoparticles can serve as carriers for small and large biomolecules as well as for synthetic compounds. The nanoparticles were prepared and colloidally stabilized with either polyethyleneimine (PEI; cationic nanoparticles) or carboxymethyl cellulose (CMC; anionic nanoparticles) and loaded with defined amounts of the fluorescently labelled proteins HTRA1, HTRA2, and BSA. The nanoparticles were purified by ultracentrifugation and characterized by dynamic light scattering and scanning electron microscopy. Various cell types (HeLa, MG-63, THP-1, and hMSC) were incubated with fluorescently labelled proteins alone or with protein-loaded cationic and anionic nanoparticles. The cellular uptake was followed by light and fluorescence microscopy, confocal laser scanning microscopy (CLSM), and flow cytometry. All proteins were readily transported into the cells by cationic calcium phosphate nanoparticles. Notably, only HTRA1 was able to penetrate the cell membrane of MG-63 cells in dissolved form. However, the application of endocytosis inhibitors revealed that the uptake pathway was different for dissolved HTRA1 and HTRA1-loaded nanoparticles. PMID:28326227

  19. The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid.

    Science.gov (United States)

    Taffet, G E; Pham, T T; Bick, D L; Entman, M L; Pownall, H J; Bick, R J

    1993-01-01

    Small amounts of dietary n-3 fatty acids can have dramatic physiological effects, including the reduction of plasma triglycerides and an elevation of cellular eicosapentanoic (EPA) and docosahexanoic acids (DHA) at the expense of arachidonic acid (AA). We investigated the effects of alterations in the fatty acid compositions of cardiac sarcoplasmic reticulum (CSR) produced by dietary manipulation on the calcium pump protein that is required for energy dependent calcium transport. CSR was isolated from rats fed menhaden oil, which is rich in n-3 fatty acids, and from control animals that were given corn oil. Relative to control membranes, those isolated from rats fed menhaden oil, had a lower content of saturated phospholipids, an increased DHA/AA ratio, and an increased ratio of n-3 to n-6 fatty acids. These changes were associated with a 30% decrease in oxalate-facilitated, ATP-dependent calcium uptake and concomitant decreased Ca-ATPase activity in the membranes from the animals fed menhaden oil. In contrast, there was no alteration in active pump sites as measured by phosphoenzyme formation. Thus, the CSR Ca-ATPase function can be altered by dietary interventions that change the composition, and possibly structure, of the phospholipid membranes thereby affecting enzyme turnover.

  20. Effect of calcium, tannic acid, phytic acid and pectin over iron uptake in an in vitro Caco-2 cell model.

    Science.gov (United States)

    Andrews, M; Briones, L; Jaramillo, A; Pizarro, F; Arredondo, M

    2014-04-01

    Calcium, phytic acid, polyphenols and fiber are major inhibitors of iron absorption and they could be found in excess in some diets, thereby altering or modifying the iron nutrition status. The purpose of this study is to evaluate the effect of calcium, tannic acid, phytic acid, and pectin over iron uptake, using an in vitro model of epithelial cells (Caco-2 cell line). Caco-2 cells were incubated with iron (10-30 μM) with or without CaCl2 (500 and 1,000 μM) for 24 h. Then, cells were challenged with phytic acid (50-150 μM); pectin (50-150 nM) or tannic acid (100-500 μM) for another 24 h. Finally, (55)Fe (10 μM) uptake was determined. Iron dialyzability was studied using an in vitro digestion method. Iron uptake in cells pre-incubated with 20 and 30 μM Fe was inhibited by CaCl2 (500 μM). Iron uptake decreased in cells cultured with tannic acid (300 μM) and CaCl2 (500-1,000 μM) (two-way ANOVA, p = 0.002). Phytic acid also decreased iron uptake mainly when cells were treated with CaCl2 (1,000 μM) (two-way ANOVA; p phytic or tannic acid (T test p Phytic acid combined with calcium is a strong iron uptake inhibitor. Pectin slightly decreased iron uptake with or without calcium. Tannic acid showed an unexpected behavior, inducing an increase on iron uptake, despite its low Fe dialyzability.

  1. Heterogeneous uptake and reactivity of formic acid on calcium carbonate particles: a Knudsen cell reactor, FTIR and SEM study.

    Science.gov (United States)

    Al-Hosney, Hashim A; Carlos-Cuellar, Sofia; Baltrusaitis, Jonas; Grassian, Vicki H

    2005-10-21

    The heterogeneous uptake and reactivity of formic acid (HCOOH), a common gas-phase organic acid found in the environment, on calcium carbonate (CaCO(3)) particles have been investigated using a Knudsen cell reactor, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). FTIR measurements show that the adsorption of formic acid on the surface of calcium carbonate results in the formation of calcium formate. Besides calcium formate, carbonic acid is also a reaction product under dry conditions (dry conditions and at low pressures, the initial uptake coefficient of formic acid on CaCO(3) particles is measured to be 3 +/- 1 x 10(-3) and decreases as the surface saturates with adsorbed products. The maximum surface coverage of formic acid under dry conditions is determined to be (3 +/- 1)x 10(14) molecules cm(-2). Under humidified conditions (RH >10%), adsorbed water on the surface of the carbonate particles participates in the surface reactivity of these particles, which results in the enhanced uptake kinetics and extent of reaction of this organic acid on CaCO(3) as well as opens up several new reaction pathways. These reaction pathways include: (i) the water-assisted dissociation of carbonic acid to CO(2) and H(2)O and (ii) the formation of calcium formate islands and crystallites, as evident by SEM images. The results presented here show that adsorbed water plays a potentially important role in the surface chemistry of gas-phase organic acids on calcium carbonate particles.

  2. Prediction of coronary artery calcium progression by FDG uptake of large arteries in asymptomatic individuals

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Sang-Geon; Park, Ki Seong; Kim, Jahae; Song, Ho-Chun [Chonnam National University Hospital, Department of Nuclear Medicine, Gwang-ju (Korea, Republic of); Kang, Sae-Ryung; Kwon, Seong Young; Jabin, Zeenat; Kim, Young Jae; Jeong, Geum-Cheol; Song, Minchul; Min, Jung-Joon; Bom, Hee-Seung [Chonnam National University Hwasun Hospital, Department of Nuclear Medicine, Hwasun-gun, Jeollanam-do (Korea, Republic of); Seon, Hyun Ju [Chonnam National University Hwasun Hospital, Department of Radiology, Hwasun-gun, Jeollanam-do (Korea, Republic of)

    2017-01-15

    The purpose of this study is to evaluate whether fluorodeoxyglucose (FDG) uptake of the large arteries can predict coronary artery calcium (CAC) progression in asymptomatic individuals. Ninety-six asymptomatic individuals who underwent FDG positron emission tomography (PET) and CAC scoring on the same day for health screening and follow-up CAC scoring ≥1 year after baseline studies (mean 4.3 years) were included. Vascular FDG uptake was measured and corrected for blood pool activity to obtain peak and average target-to-blood pool ratios (TBRpeak and TBRavg, respectively) for the carotid arteries, and ascending and abdominal aorta. CAC scores at baseline and follow-up of each individual were measured and absolute CAC change (ΔCAC), annual CAC change (ΔCAC/year), and annual CAC change rate (ΔCAC%/year) were calculated. CAC progression was defined as ΔCAC >0 for individuals with negative baseline CAC; ΔCAC/year ≥10 for those with baseline CAC of 0uptake and other clinical risk factors were compared between CAC-progressors and non-CAC-progressors. Multivariate analysis was performed to evaluate whether vascular FDG uptake can independently predict CAC progression. Thirty-one subjects showed CAC progression. CAC-progressors showed significantly higher TBRpeak and TBRavg as compared to non-CAC-progressors for all three arteries. TBRpeak of the abdominal aorta was significantly associated with CAC progression in multivariate analysis, with age and baseline CAC. A higher TBRpeak of the abdominal aorta (≥2.11) was associated with CAC progression among subjects with negative baseline CAC only. In subjects with positive baseline CAC, only the amount of baseline CAC was significantly associated with CAC progression. However, the positive predictive value of the TBRpeak of the abdominal aorta was <40 % when age was <58 or baseline CAC was negative. Higher FDG uptake of the large arteries is

  3. Effect of flunarizine and calcium on serotonin uptake in human and rat blood platelets and rat synaptosomes

    DEFF Research Database (Denmark)

    Jensen, P N; Smith, D F; Poulsen, J H

    1994-01-01

    in blood platelets, whereas no effect was observed in synaptosomes. Flunarizine inhibited serotonin uptake in a concentration dependent manner with an IC50 value of 1 mumol/L in blood platelets and 5 mumol/L in synaptosomes. The inhibition did not depend on the presence of extracellular calcium indicating...

  4. {sup 41}Ca as a tracer for calcium uptake and deposition in heart tissue during ischemia and reperfusion

    Energy Technology Data Exchange (ETDEWEB)

    Southon, J.R. [Lawrence Livermore National Lab., CA (United States); Bishop, M.S.; Kost, G.J. [California Univ., Davis, CA (United States). Dept. of Medical Pathology and Biomedical Engineering

    1993-09-17

    We have developed techniques and are commencing experiments using enriched {sup 41}Ca as a tracer in isolated rabbit heart preparations. The aims of the study are to measure calcium uptake and deposition in response to cardiac ischemia and reperfusion, and to investigate events and mechanism leading to irreversible myocyte injury.

  5. A high-affinity molybdate transporter in eukaryotes.

    Science.gov (United States)

    Tejada-Jiménez, Manuel; Llamas, Angel; Sanz-Luque, Emanuel; Galván, Aurora; Fernández, Emilio

    2007-12-11

    Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.

  6. Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake.

    Science.gov (United States)

    Nathan, Sarah R; Pino, Nicholas W; Arduino, Daniela M; Perocchi, Fabiana; MacMillan, Samantha N; Wilson, Justin J

    2017-03-20

    The mixed-valent oxo-bridged ruthenium complex [(HCO2)(NH3)4Ru(μ-O)Ru(NH3)4(O2CH)](3+), known as Ru360, is a selective inhibitor of mitochondrial calcium uptake. Although this compound is useful for studying the role of mitochondrial calcium in biological processes, its widespread availability is limited because of challenges in purification and characterization. Here, we describe our investigations of three different synthetic methods for the preparation of a functional analogue of this valuable compound. We demonstrate that this analogue, isolated from our procedures, exhibits potent mitochondrial calcium uptake inhibitory properties in permeabilized HeLa cells and in isolated mitochondria.

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

    Science.gov (United States)

    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.

  8. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  9. AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae.

    Science.gov (United States)

    Aouida, Mustapha; Leduc, Anick; Poulin, Richard; Ramotar, Dindial

    2005-06-24

    Polyamines play essential functions in many aspects of cell biology. Plasma membrane transport systems for the specific uptake of polyamines exist in most eukaryotic cells but have been very recently identified at the molecular level only in the parasite Leishmania. We now report that the high affinity polyamine permease in Saccharomyces cerevisiae is identical to Agp2p, a member of the yeast amino acid transporter family that was previously identified as a carnitine transporter. Deletion of AGP2 dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines, and transformation with an AGP2 expression vector restored polyamine transport in agp2delta mutants. Yeast mutants deficient in polyamine biosynthesis required >10-fold higher concentrations of exogenous putrescine to restore cell proliferation upon deletion of the AGP2 gene. Disruption of END3, a gene required for an early step of endocytosis, increased the abundance of Agp2p, an effect that was paralleled by a marked up-regulation of spermidine transport velocity. Thus, AGP2 encodes the first eukaryotic permease that preferentially uses spermidine over putrescine as a high affinity substrate and plays a central role in the uptake of polyamines in yeast.

  10. Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons

    Science.gov (United States)

    Mohamed, Habib A.; Mosier, Dennis R.; Zou, Ling L.; Siklos, Laszlo; Alexianu, Maria E.; Engelhardt, Jozsef I.; Beers, David R.; Le, Wei-dong; Appel, Stanley H.

    2002-01-01

    Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals. Copyright 2002 Wiley-Liss, Inc.

  11. High affinity retinoic acid receptor antagonists: analogs of AGN 193109.

    Science.gov (United States)

    Johnson, A T; Wang, L; Gillett, S J; Chandraratna, R A

    1999-02-22

    A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated.

  12. High affinity ligands for 'diazepam-insensitive' benzodiazepine receptors.

    Science.gov (United States)

    Wong, G; Skolnick, P

    1992-01-14

    Structurally diverse compounds have been shown to possess high affinities for benzodiazepine receptors in their 'diazepam-sensitive' (DS) conformations. In contrast, only the imidazobenzodiazepinone Ro 15-4513 has been shown to exhibit a high affinity for the 'diazepam-insensitive' (DI) conformation of benzodiazepine receptors. We examined a series of 1,4-diazepines containing one or more annelated ring systems for their affinities at DI and DS benzodiazepine receptors, several 1,4-diazepinone carboxylates including Ro 19-4603, Ro 16-6028 and Ro 15-3505 were found to possess high affinities (Ki approximately 2.6-20 nM) for DI. Nonetheless, among the ligands examined, Ro 15-4513 was the only substance with a DI/DS potency ratio approximately 1; other substances had ratios ranging from 13 to greater than 1000. Ligands with high to moderate affinities at DI were previously classified as partial agonists, antagonists, or partial inverse agonists at DS benzodiazepine receptors, but behaved as 'GABA neutral' (antagonist) substances at DI. The identification of several additional high affinity ligands at DI benzodiazepine receptors may be helpful in elucidating the pharmacological and physiological importance of these sites.

  13. Increased strontium uptake in trabecular bone of ovariectomized calcium-deficient rats treated with strontium ranelate or strontium chloride.

    Science.gov (United States)

    Pemmer, Bernhard; Hofstaetter, Jochen G; Meirer, Florian; Smolek, Stephan; Wobrauschek, Peter; Simon, Rolf; Fuchs, Robyn K; Allen, Matthew R; Condon, Keith W; Reinwald, Susan; Phipps, Roger J; Burr, David B; Paschalis, Eleftherios P; Klaushofer, Klaus; Streli, Christina; Roschger, Paul

    2011-11-01

    Based on clinical trials showing the efficacy to reduce vertebral and non-vertebral fractures, strontium ranelate (SrR) has been approved in several countries for the treatment of postmenopausal osteoporosis. Hence, it is of special clinical interest to elucidate how the Sr uptake is influenced by dietary Ca deficiency as well as by the formula of Sr administration, SrR versus strontium chloride (SrCl(2)). Three-month-old ovariectomized rats were treated for 90 days with doses of 25 mg kg(-1) d(-1) and 150 mg kg(-1) d(-1) of SrR or SrCl(2) at low (0.1% Ca) or normal (1.19% Ca) Ca diet. Vertebral bone tissue was analysed by confocal synchrotron-radiation-induced micro X-ray fluorescence and by backscattered electron imaging. Principal component analysis and k-means clustering of the acquired elemental maps of Ca and Sr revealed that the newly formed bone exhibited the highest Sr fractions and that low Ca diet increased the Sr uptake by a factor of three to four. Furthermore, Sr uptake in bone of the SrCl(2)-treated animals was generally lower compared with SrR. The study clearly shows that inadequate nutritional calcium intake significantly increases uptake of Sr in serum as well as in trabecular bone matrix. This indicates that nutritional calcium intake as well as serum Ca levels are important regulators of any Sr treatment.

  14. 1α,25-Dihydroxyvitamin D(3) signaling pathways on calcium uptake in 30-day-old rat Sertoli cells.

    Science.gov (United States)

    Zanatta, Leila; Zamoner, Ariane; Gonçalves, Renata; Zanatta, Ana Paula; Bouraïma-Lelong, Hélène; Carreau, Serge; Silva, Fátima Regina Mena Barreto

    2011-11-29

    1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.

  15. Calcium Uptake via Mitochondrial Uniporter Contributes to Palmitic Acid-induced Apoptosis in Mouse Podocytes.

    Science.gov (United States)

    Yuan, Zeting; Cao, Aili; Liu, Hua; Guo, Henjiang; Zang, Yingjun; Wang, Yi; Wang, Yunman; Wang, Hao; Yin, Peihao; Peng, Wen

    2017-02-09

    Podocytes are component cells of the glomerular filtration barrier, and their loss by apoptosis is the main cause of proteinuria that leads to diabetic nephropathy (DN). Therefore, insights into podocyte apoptosis mechanism would allow a better understanding of DN pathogenesis and thus help develop adequate therapeutic strategies. Here, we investigated the molecular mechanism of palmitic acid-inhibited cell death in mouse podocytes, and found that palmitic acid increased cell death in a dose- and time-dependent manner. Palmitic acid induces apoptosis in podocytes through up-regulation of cytosolic and mitochondrial Ca(2+) , mitochondrial membrane potential (MMP), cytochrome c release and depletion of endoplasmic reticulum (ER) Ca(2+) , The intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N, N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), partially prevented this up-regulation whereas 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor (IP3R) inhibitor; dantrolene, a ryanodine receptor (RyR) inhibitor; and 4,4'-diisothiocyanatostibene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, had no effect. Interestingly, ruthenium red and Ru360, both inhibitors of the mitochondrial Ca(2+) uniporter (MCU), blocked palmitic acid-induced mitochondrial Ca(2+) elevation, cytochrome c release from mitochondria to cytosol, and apoptosis. siRNA to MCU markedly reduced curcumin-induced apoptosis. These data indicate that Ca(2+) uptake via mitochondrial uniporter contributes to palmitic acid-induced apoptosis in mouse podocytes. This article is protected by copyright. All rights reserved.

  16. Effect of sepsis on calcium uptake and content in skeletal muscle and regulation in vitro by calcium of total and myofibrillar protein breakdown in control and septic muscle: Results from a preliminary study

    Energy Technology Data Exchange (ETDEWEB)

    Benson, D.W.; Hasselgren, P.O.; Hiyama, D.T.; James, J.H.; Li, S.; Rigel, D.F.; Fischer, J.E.

    1989-07-01

    Because high calcium concentration in vitro stimulates muscle proteolysis, calcium has been implicated in the pathogenesis of increased muscle breakdown in different catabolic conditions. Protein breakdown in skeletal muscle is increased during sepsis, but the effect of sepsis on muscle calcium uptake and content is not known. In this study the influence of sepsis, induced in rats by cecal ligation and puncture, on muscle calcium uptake and content was studied. Sixteen hours after cecal ligation and puncture or sham operation, calcium content of the extensor digitorum longus (EDL) and soleus (SOL) muscles was determined with an atomic absorption spectrometer. Calcium uptake was measured in intact SOL muscles incubated in the presence of calcium 45 (45Ca) for between 1 and 120 minutes. Total and myofibrillar protein breakdown was determined in SOL muscles, incubated in the presence of different calcium concentrations (0; 2.5; 5.0 mmol/L), and measured as release into the incubation medium of tyrosine and 3-methylhistidine (3-MH), respectively. Calcium content was increased by 51% (p less than 0.001) during sepsis in SOL and by 10% (p less than 0.05) in EDL muscle. There was no difference in 45Ca uptake between control and septic muscles during the early phase (1 to 5 minutes) of incubation. During more extended incubation (30 to 120 minutes), muscles from septic rats took up significantly more 45Ca than control muscles (p less than 0.05). Tyrosine release by incubated SOL muscles from control and septic rats was increased when calcium was added to the incubation medium, and at a calcium concentration of 2.5 mmol/L, the increase in tyrosine release was greater in septic than in control muscle. Addition of calcium to the incubation medium did not affect 3-MH release in control or septic muscle.

  17. Rapid changes in skeletal muscle calcium uptake induced in vitro by 1,25-dihydroxyvitamin D/sub 3/ are suppressed by calcium channel blockers

    Energy Technology Data Exchange (ETDEWEB)

    de Boland, A.R.; Boland, R.L.

    1987-05-01

    Previous investigations have shown that 1,25-dihydroxyvitamin D/sub 3/ (1,25-(OH)/sub 2/D/sub 3/) stimulates muscle Ca uptake through a nuclear mechanism. The possibility that 1,25-(OH)/sub 2/D/sub 3/ would induce rapid changes in muscle Ca fluxes independent of de novo protein synthesis was investigated in the present work. In vitro preparations of soleus muscles obtained from vitamin D-deficient chicks were used. A significant increase in /sup 45/Ca labeling of the tissue was already observed after 3-min treatment with 2.4 X 10(-10) M 1,25-(OH)/sub 2/D/sub 3/. This early stimulation in muscle Ca uptake became maximal at 10-15 min. Cycloheximide (50 microM) did not block the effect of the metabolite at 15 and 30 min. However, the antibiotic effectively blocked the increase in Ca uptake induced by 1,25-(OH)/sub 2/D/sub 3/ after 1-h treatment. The rapid 1,25-(OH)/sub 2/D/sub 3/-dependent stimulation of /sup 45/Ca labeling of soleus muscle was not associated to changes in lipid synthesis as assessed by measurements of /sup 3/H-glycerol incorporation into the tissue lipids. However, the calcium antagonists verapamil and nifedipine (50 microM) abolished the stimulation in Ca uptake produced by 1,25-(OH)/sub 2/D/sub 3/ in 5 min. These results suggest that 1,25-(OH)/sub 2/D/sub 3/ can act directly at the muscle membrane level affecting Ca fluxes through Ca channels.

  18. A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

    Science.gov (United States)

    Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

    2014-11-01

    Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  2. High affinity binding site-mediated prevention of chemical absorption across the gastrointestinal tract.

    Science.gov (United States)

    Rasmussen, M V; Barker, T T; Silbart, L K

    2001-12-15

    Preventing mucosal absorption of low-molecular weight compounds such as carcinogens, toxins and drugs could help prevent many diseases. To characterize the effects of dose and timing on high-affinity binding site mediated sequestration of specific chemical ligands in the gastrointestinal tract, avidin was perorally-administered to mice either prior to or mixed with 3H-biotin. Avidin enhanced fecal 3H-biotin excretion in a dose-dependent manner, consistent with the accepted mechanism of egg white-induced biotin deficiency syndrome. Avidin administration up to 4 h before 3H-biotin administration also enhanced fecal 3H-biotin excretion. Activated charcoal (AC) reduced 3H-biotin absorption when mixed with 3H-biotin before ingestion, but was ineffective when ingested prior to 3H-biotin. These studies suggest that ingestion of high-affinity protein binding sites can establish an absorptive barrier at the gastrointestinal mucosa to prevent the uptake of unwanted low molecular-weight chemicals.

  3. Cellular uptake of {sup 99m}TcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Guillermet, Stephanie; Vuillez, Jean-Philippe; Caravel, Jean-Pierre; Marti-Batlle, Daniele; Fagret, Daniel [Universite de Grenoble, Radiopharmaceutiques Biocliniques, La Tronche (France); Fontaine, Eric [Universite de Grenoble, Laboratoire de Bioenergetique Fondamentale et Appliquee, Grenoble (France); Pasqualini, Roberto [Cis Bio International Schering SA, Gif-sur-Yvette (France)

    2006-01-01

    A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ({sup 99m}TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether {sup 99m}TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of {sup 99m}TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of {sup 99m}TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG{sub 2}M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of {sup 99m}TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of {sup 99m}TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG{sub 2}M. The uptake of {sup 99m}TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that {sup 99m}TcN-NOET might be a marker of cell proliferation. (orig.)

  4. Influence of external calcium and thapsigargin on the uptake of polystyrene beads by the macrophage-like cell lines U937 and MH-S

    Science.gov (United States)

    2014-01-01

    Background Macrophages are equipped with several receptors for the recognition of foreign particles and pathogens. Upon binding to these receptors, particles become internalized. An interaction of particles with macrophage surface receptors is accompanied by an increase in cytosolic calcium concentration. This calcium is provided by intracellular stores and also by an influx of external calcium upon activation of the calcium channels. Nevertheless, the role of calcium in phagocytosis remains controversial. Some researchers postulate the necessity of calcium in Fc-receptor-mediated phagocytosis and a calcium-independent phagocytosis of complement opsonized particles. Others refute the need for calcium in Fc-receptor-mediated phagocytosis by macrophages. Methods In this study, the influence of external calcium concentrations and thapsigargin on the phagocytosis of polystyrene latex beads by the macrophage-like cell lines MH-S (murine) and differentiated U937 (human) was analyzed. The phagocytosis efficiency was determined by flow cytometry and was evaluated statistically by ANOVA test and Dunett’s significance test, or ANOVA and Bonferroni’s Multiple Comparison. Results Acquired data revealed an external calcium-independent way of internalization of non-functionalized polystyrene latex beads at free calcium concentrations ranging from 0 mM to 3 mM. The phagocytosis efficiency of the cells is not significantly decreased by a complete lack of external calcium. Furthermore, the presence of thapsigargin, known to lead to an increase of cytosolic calcium levels, did not have a significant enhancing influence on bead uptake by MH-S cells and only an enhancing effect on bead uptake by macrophage-like U937 cells at an external calcium concentration of 4 mM. Conclusion The calcium-independent phagocytosis process and the decrease of phagocytosis efficiency in the presence of complement receptor inhibitor staurosporine lead to the assumption that besides other calcium

  5. Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling.

    Science.gov (United States)

    Yi, Jianxun; Ma, Changling; Li, Yan; Weisleder, Noah; Ríos, Eduardo; Ma, Jianjie; Zhou, Jingsong

    2011-09-16

    Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.

  6. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Calcium

    Science.gov (United States)

    ... in luck if you like sardines and canned salmon with bones. Almond milk. previous continue Working Calcium ... drinks, and cereals. Other Considerations for Building Bones Vitamin D is essential for calcium absorption, so it's ...

  8. Study on Characteristics of Calcium Uptake by Young Fruit of Apple (Malus pumila) and Its Regulation by Hormone

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Field trial, pot experiment with 45Ca tracer, plant analysis were used to investigate the characteristics of calcium uptake by young fruit of apple and its regulation by IAA, GA and NAA. The results indicated that calcium should be applied directly on the surface of young fruits because calcium applied on leaves could be hardly transfered to fruits. The proper Ca applying period was the first month of young fruits formation, and the proper concentration of CaCl2 applied was 0. 5%. Applying Ca directly on the surface of young fruits could increase the weight and quality of fruits. The process of transfering Ca2+ from fruit surface into pulp tissue could be accelerated by IAA, GA or NAA, which also led to an increment on 2% HOAc extractable Ca. Meanwhile, the Ca existed in the stalk and leaves could be strongly transported into fruits by applying IAA on the fruit surface, resulting in too much accumulation of Ca in fruit and bad quality of fruit, while no such sighs were observed with GA or NAA.

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

    Science.gov (United States)

    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

  10. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    Directory of Open Access Journals (Sweden)

    Josep V Forment

    Full Text Available 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.

  11. TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via Different Routes is Calcium-Dependent.

    Science.gov (United States)

    Tan, Ling-Yan; Huang, Bin; Xu, Shen; Wei, Zhong-Bo; Yang, Liu-Yan; Miao, Ai-Jun

    2016-07-19

    Calcium plays versatile roles in aquatic ecosystems. In this study, we investigated its effects on the uptake of polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) by the water flea (cladoceran) Daphnia magna. Particle distribution in these daphnids was also visualized using synchrotron radiation-based micro X-ray fluorescence spectroscopy, transmission electron microscopy, and scanning electron microscopy. At low ambient Ca concentrations in the experimental medium ([Ca]dis), PAA-TiO2-NPs were well dispersed and distributed throughout the daphnid; the particle concentration was highest in the abdominal zone and the gut, as a result of endocytosis and passive drinking of the nanoparticles, respectively. Further, Ca induced PAA-TiO2-NP uptake as a result of the increased Ca influx. At a high [Ca]dis, the PAA-TiO2-NPs formed micrometer-sized aggregates that were ingested by D. magna and concentrated only in its gut, independent of the Ca influx. Our results demonstrated the multiple effects of Ca on nanoparticle bioaccumulation. Specifically, well-dispersed nanoparticles were taken up by D. magna through endocytosis and passive drinking whereas the uptake of micrometer-sized aggregates relied on active ingestion.

  12. New insights in the structure and biology of the high affinity receptor for IgE (Fc epsilon RI) on human epidermal Langerhans cells.

    Science.gov (United States)

    Bieber, T; Kraft, S; Jürgens, M; Strobel, I; Haberstok, J; Tomov, H; Regele, D; de la Salle, H; Wollenberg, A; Hanau, D

    1996-10-01

    The recent structural and functional analysis of the high affinity receptor for IgE (Fc epsilon RI) expressed on human epidermal Langerhans cells (LC) revealed new aspects of the biology of this structure. In contrast to basophils and mast cells where this receptor seems to be expressed constitutively at a constant level, the expression of Fc epsilon RI on LC varies on the donor and the inflammatory environment of the cells and lacks the classical beta-chain. This also implies functional differences most probably related to the expression level. Although the signalling pathway seems to be similar to that of basophils or mast cells, LC from individuals with atopic dermatitis are fully activated by receptor ligation while LC from normal individuals fail to exhibit calcium mobilization under the same conditions. Finally, LC from normal and atopic individuals use Fc epsilon RI to maximize antigen uptake via specific IgE and subsequent presentation to T cells. Thus, Fc epsilon RI expressed on LC differs in terms of structure and function from that expressed on effector cells of anaphylaxis.

  13. The high-affinity immunoglobulin E receptor as pharmacological target.

    Science.gov (United States)

    Blank, Ulrich; Charles, Nicolas; Benhamou, Marc

    2016-05-05

    The high-affinity receptor for immunoglobulin E is expressed mainly on mast cells and basophils, but also on neutrophils, eosinophils, platelets, monocytes, Langerhans and dendritic cells, airway smooth muscle cells and some nerve cells. Its main function is, upon its engagement by IgE and specific antigen, to trigger a powerful defense against invading pathogens and a rapid neutralization of dangerous toxic substances introduced in the body. This powerful response could be wielded against tumors. But, when control over this receptor is lost, its unchecked activation can induce an array of diseases, some of which can lead to death. In this review we will summarize the pharmacological approaches and strategies that are currently used, or under study, to harness or wield activation of this receptor for therapeutic purposes.

  14. Inhibitory effects of calcium channel blockers on thyroid hormone uptake in neonatal rat cardiomyocytes

    NARCIS (Netherlands)

    F.A. Verhoeven; E.P.C.M. Moerings (Ellis); J.M.J. Lamers (Jos); G. Hennemann; T.J. Visser (Theo); M.E. Everts (Maria)

    2001-01-01

    textabstractThe effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C in medium with 0.5% BSA for [125I]T3 (

  15. Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts

    Science.gov (United States)

    Stagg, Amy R.; Fleming, Judith C.; Baker, Meghan A.; Sakamoto, Massayuki; Cohen, Nadine; Neufeld, Ellis J.

    1999-01-01

    We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5–14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10–30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400–550 nM; Vmax 11 pmol/min/106 cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death. J. Clin. Invest. 103:723–729 (1999). PMID:10074490

  16. Probing the limit of magnesium uptake by β-tricalcium phosphate in biphasic mixtures formed from calcium deficient apatites

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, P. Nandha; Mishra, Sandeep K.; Kannan, S., E-mail: para_kanna@yahoo.com

    2015-11-15

    A series of magnesium doped non-stoichiometric calcium deficient apatites were synthesized through an aqueous precipitation route. The resultant structural changes during heat treatment were investigated by X-ray diffraction, Raman and FT-IR spectroscopy and Rietveld refinement. The results confirmed the formation of biphasic mixtures comprising Ca{sub 10}(PO{sub 4}){sub 6}(OH){sub 2} and β-Ca{sub 3}(PO{sub 4}){sub 2} after heat treatment at 1000 °C with the preferential occupancy of Mg{sup 2+} at the crystal lattice of β-Ca{sub 3}(PO{sub 4}){sub 2}. The concentration of Mg{sup 2+} uptake in β-Ca{sub 3}(PO{sub 4}){sub 2} is limited till reaching the stoichiometric ratio of (Ca+Mg)/P=1.67 and beyond this stoichiometric value [(Ca+Mg)/P>1.67], Mg{sup 2+} precipitates as Mg(OH){sub 2} and thereafter gets converted to MgO during heat treatment. Any kind of Mg{sup 2+} uptake in the crystal lattice of Ca{sub 10}(PO{sub 4}){sub 6}(OH){sub 2} is discarded from the investigation. - Highlights: • Aqueous co-precipitation of calcium deficient apatites with excess magnesium (Mg{sup 2+}) additions. • Heat treatments beyond 800 °C results in the formation of biphasic apatite mixtures. • Mg{sup 2+} gets accommodated at the β-Ca{sub 3}(PO{sub 4}){sub 2} lattice of biphasic mixtures. • Mg{sup 2+} additions exceeding stoichiometric value (Ca/P>1.67) results in its formation as MgO. • Mg{sup 2+} occupancy at β-Ca{sub 3}(PO{sub 4}){sub 2} lattice delays its allotropic conversion α-Ca{sub 3}(PO{sub 4}){sub 2} till 1350 °C.

  17. [Experiments on the mechanism of action of vascular spasmolytics. 4. Effect of nitroprusside sodium, nitroglycerin, prenylamine and verapamil on the calcium uptake of microsomes of the smooth bascular muscles].

    Science.gov (United States)

    Klinner, U; Ehlers, D; Fermum, R; Meisel, P

    1977-01-01

    Nitroprusside-sodium, nitroglycerol, and verapamil had no effect on the calcium uptake by microsomes from the carotid artery of cattle. Prenylamine reduced the passive binding and the active uptake and released already bound calcium. The basal Mg-dependendent ATPase and Ca-stimulatable Mg-ATPase were inhibited by prenylamine.

  18. Hyperosmolar sodium chloride is toxic to cultured neurons and causes reduction of glucose metabolism and ATP levels, an increase in glutamate uptake, and a reduction in cytosolic calcium.

    Science.gov (United States)

    Morland, Cecilie; Pettersen, Mi Nguyen; Hassel, Bjørnar

    2016-05-01

    Elevation of serum sodium, hypernatremia, which may occur during dehydration or treatment with sodium chloride, may cause brain dysfunction and damage, but toxic mechanisms are poorly understood. We found that exposure to excess NaCl, 10-100mmol/L, for 20h caused cell death in cultured cerebellar granule cells (neurons). Toxicity was due to Na(+), since substituting excess Na(+) with choline reduced cell death to control levels, whereas gluconate instead of excess Cl(-) did not. Prior to cell death from hyperosmolar NaCl, glucose consumption and lactate formation were reduced, and intracellular aspartate levels were elevated, consistent with reduced glycolysis or glucose uptake. Concomitantly, the level of ATP became reduced. Pyruvate, 10mmol/L, reduced NaCl-induced cell death. The extracellular levels of glutamate, taurine, and GABA were concentration-dependently reduced by excess NaCl; high-affinity glutamate uptake increased. High extracellular [Na(+)] caused reduction in intracellular free [Ca(2+)], but a similar effect was seen with mannitol, which was not neurotoxic. We suggest that inhibition of glucose metabolism with ensuing loss of ATP is a neurotoxic mechanism of hyperosmolar sodium, whereas increased uptake of extracellular neuroactive amino acids and reduced intracellular [Ca(2+)] may, if they occur in vivo, contribute to the cerebral dysfunction and delirium described in hypernatremia.

  19. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    Science.gov (United States)

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-07

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

  20. Development and characterization of high affinity leptins and leptin antagonists.

    Science.gov (United States)

    Shpilman, Michal; Niv-Spector, Leonora; Katz, Meirav; Varol, Chen; Solomon, Gili; Ayalon-Soffer, Michal; Boder, Eric; Halpern, Zamir; Elinav, Eran; Gertler, Arieh

    2011-02-11

    Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

  1. Development and Characterization of High Affinity Leptins and Leptin Antagonists*

    Science.gov (United States)

    Shpilman, Michal; Niv-Spector, Leonora; Katz, Meirav; Varol, Chen; Solomon, Gili; Ayalon-Soffer, Michal; Boder, Eric; Halpern, Zamir; Elinav, Eran; Gertler, Arieh

    2011-01-01

    Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin. PMID:21119198

  2. Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Jun-Yi Wang; Yong-Guan Zhu; Qi-Rong Shen; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2008-01-01

    Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.

  3. TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells.

    Science.gov (United States)

    Mitrovic, Sandra; Nogueira, Cristina; Cantero-Recasens, Gerard; Kiefer, Kerstin; Fernández-Fernández, José M; Popoff, Jean-François; Casano, Laetitia; Bard, Frederic A; Gomez, Raul; Valverde, Miguel A; Malhotra, Vivek

    2013-05-28

    Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion. DOI:http://dx.doi.org/10.7554/eLife.00658.001.

  4. Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

    Science.gov (United States)

    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. PMID:23382943

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

    Directory of Open Access Journals (Sweden)

    Shahin S Ali

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Graham S Baldwin

    Full Text Available 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.

  7. PREPARATION AND CHARACTERISTICS OF ANIONIC POLYACRYLAMIDES CONTAINING DIRECT DYE WITH A HIGH AFFINITY FOR CELLULOSE

    Directory of Open Access Journals (Sweden)

    Shingo Yokota

    2009-05-01

    Full Text Available Direct dye with a high affinity for cellulose substrate was utilized as a cellulose anchor to promote retention of paper strengthening additives under various conditions associated with the wet end of a paper machine. Direct Red 28 (DR was covalently linked to anionic polyacrylamide (A-PAM via a condensation reaction using water-soluble carbodiimide. The DR-conjugated A-PAM (DR-A-PAM demonstrated good retention efficiency, resulting in strength enhancement of handsheets. Anionic trash showed no interference with the performance of DR-A-PAM in the wet end, while the additive performance was sensitive to calcium ions. Surface plasmon resonance analysis gave useful information on the cellulose-anchoring ability of DR-A-PAM. Dye molecules were irreversibly adsorbed onto the cellulose substrate under aqueous conditions, while A-PAM possessed no significant affinity for cellulose. These results suggest that anionic DR moieties in DR-A-PAM molecules served as a cellulose-anchor, possibly due to multiple CH-π interaction between hydrophobic face of cellulose substrate and π-conjugated system of dye molecules. Such a unique interaction of direct dye and cellulose provides a new insight into the wet end system, and does not depend on conventional electrostatic attraction.

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

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    2013-01-01

    Full Text Available 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.

  9. The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron.

    Science.gov (United States)

    Miethke, Marcus; Monteferrante, Carmine G; Marahiel, Mohamed A; van Dijl, Jan Maarten

    2013-10-01

    Efficient uptake of iron is of critical importance for growth and viability of microbial cells. Nevertheless, several mechanisms for iron uptake are not yet clearly defined. Here we report that the widely conserved transporter EfeUOB employs an unprecedented dual-mode mechanism for acquisition of ferrous (Fe[II]) and ferric (Fe[III]) iron in the bacterium Bacillus subtilis. We show that the binding protein EfeO and the permease EfeU form a minimal complex for ferric iron uptake. The third component EfeB is a hemoprotein that oxidizes ferrous iron to ferric iron for uptake by EfeUO. Accordingly, EfeB promotes growth under microaerobic conditions where ferrous iron is more abundant. Notably, EfeB also fulfills a vital role in cell envelope stress protection by eliminating reactive oxygen species that accumulate in the presence of ferrous iron. In conclusion, the EfeUOB system contributes to the high-affinity uptake of iron that is available in two different oxidation states. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Reciprocal enhancement of uptake and toxicity of cadmium and calcium in rainbow trout (Oncorhynchus mykiss) liver mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Adiele, Reginald C.; Stevens, Don [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3 (Canada); Kamunde, Collins, E-mail: ckamunde@upei.ca [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3 (Canada)

    2010-03-01

    The interactive effects of cadmium (Cd) and calcium (Ca) on energy metabolism in rainbow trout liver mitochondria were studied to test the prediction that Ca would protect against Cd-induced mitochondrial liability. Isolated rainbow trout liver mitochondria were energized with malate and glutamate and exposed to increasing concentrations (5-100 {mu}M) of Cd and Ca singly and in combination at 15 {sup o}C. Accumulation of Cd and Ca in the mitochondria and mitochondrial respiration (oxygen consumption) rates were measured. Additionally, un-energized mitochondria were incubated with low doses (1 {mu}M) of Cd and Ca singly and in combination, with time-course measurements of cation accumulation/binding and oxygen consumption rates. In energized actively phosphorylating mitochondria, the uptake rates of both Cd and Ca were dose-dependent and enhanced when administered concurrently. Upon low-dose incubation, Cd accumulation was rapid and peaked in 5 min, while no appreciable uptake of Ca occurred. Functionally, the resting (state 4, ADP-limited) respiration rate was not affected in the dose-response exposure, but it decreased remarkably on low-dose incubation. Adenosine diphosphate (ADP)-stimulated respiration (state 3) rate was impaired dose-dependently with maximal inhibitions (at the highest dose, 100 {mu}M) of 32, 64 and 73% for Ca, Cd, and combined exposures, respectively. The combined effects of Ca and Cd suggested synergistic (more than additive) action and partial additivity of effects at low and higher doses of the two cations, respectively. Moreover, on a molar basis, Cd was twice as toxic as Ca to rainbow trout liver mitochondria and when combined, approximately 90% of the effects were attributable to Cd. The coupling efficiency, as measured by respiratory control ratio (RCR) and phosphorylation efficiency, measured as ADP/O ratio, both decreased as the exposure dosage and duration increased. In addition, Cd and Ca exposure decreased mitochondrial proton leak

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  13. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites.

    Science.gov (United States)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina; Vogensen, Stine B; Lehel, Szabolcs; Thiesen, Louise; Bundgaard, Christoffer; Clausen, Rasmus P; Knudsen, Gitte M; Herth, Matthias M; Wellendorph, Petrine; Frølund, Bente

    2017-01-18

    γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report the (11)C-labeling and subsequent evaluation of [(11)C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative autoradiography on sections of pig brain was performed using [(3)H]HOCPCA. In vivo evaluation of [(11)C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [(11)C]HOCPCA.

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

    Science.gov (United States)

    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.

  15. A KDP-LIKE, HIGH-AFFINITY, K+-TRANSLOCATING ATPASE IS EXPRESSED DURING GROWTH OF RHODOBACTER-SPHAEROIDES IN LOW POTASSIUM MEDIA - DISTRIBUTION OF THIS K+-ATPASE AMONG PURPLE NONSULFUR PHOTOTROPHIC BACTERIA

    NARCIS (Netherlands)

    ABEE, T; HELLINGWERF, KJ; BAKKER, EP; SIEBERS, A; KONINGS, WN

    1992-01-01

    Cells of the purple non-sulphur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. Antibodies against the catalytic KdpB protein or the whole KdpABC complex of Escherichia coli cross-react with a 70.0 kDa R. sphaeroides protein

  16. Twins in spirit part II: DOTATATE and high-affinity DOTATATE - the clinical experience

    Energy Technology Data Exchange (ETDEWEB)

    Brogsitter, Claudia; Zoephel, Klaus; Hartmann, Holger; Kotzerke, Joerg [Technische Universitaet Dresden, Department of Nuclear Medicine, Dresden (Germany); Schottelius, Margret; Wester, Hans-Juergen [Technische Universitaet Muenchen, Pharmaceutical Radiochemistry and Department of Nuclear Medicine, Muenchen (Germany)

    2014-06-15

    Over recent decades interest in diagnosis and treatment of neuroendocrine tumours (NET) has steadily grown. The basis for diagnosis and therapy of NET with radiolabelled somatostatin (hsst) analogues is the variable overexpression of hsst receptors (hsst1-5 receptors). We hypothesized that radiometal derivatives of DOTA-iodo-Tyr{sup 3}-octreotide analogues might be excellent candidates for somatostatin receptor imaging. We therefore explored the diagnostic potential of {sup 68}Ga-DOTA-iodo-Tyr{sup 3}-octreotate [{sup 68}Ga-DOTA,3-iodo-Tyr{sup 3},Thr{sup 8}]octreotide ({sup 68}Ga-HA-DOTATATE; HA, high-affinity) compared to the established {sup 68}Ga-DOTA-Tyr{sup 3}-octreotate ({sup 68}Ga-DOTATATE) in vivo. The study included 23 patients with known somatostatin receptor-positive metastases from NETs, thyroid cancer or glomus tumours who were investigated with both {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE. A patient-based and a lesion-based comparative analysis was carried out of normal tissue distribution and lesion detectability in a qualitative and a semiquantitative manner. {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE showed comparable uptake in the liver (SUV{sub mean} 8.9 ± 2.2 vs. 9.3 ± 2.5, n.s.), renal cortex (SUV{sub mean} 13.3 ± 3.9 vs. 14.5 ± 3.7, n.s.) and spleen (SUV{sub mean} 24.0 ± 6.7 vs. 22.9 ± 7.3, n.s.). A somewhat higher pituitary uptake was found with {sup 68}Ga-HA-DOTATATE (SUV{sub mean} 6.3 ± 1.8 vs. 5.4 ± 2.1, p < 0.05). On a lesion-by-lesion basis a total of 344 lesions were detected. {sup 68}Ga-HA-DOTATATE demonstrated 328 lesions (95.3 % of total lesions seen), and {sup 68}Ga-DOTATATE demonstrated 332 lesions (96.4 %). The mean SUV{sub max} of all lesions was not significantly different between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE (17.8 ± 11.4 vs. 16.7 ± 10.7, n.s.). Our analysis demonstrated very good concordance between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE PET data. As the availability and use of {sup

  17. Uptake and Transport of Calcium in Plants%植物钙素吸收和运转

    Institute of Scientific and Technical Information of China (English)

    杨洪强; 接玉玲

    2005-01-01

    近年来,钙素在植物体内的吸收和运输研究主要集中在细胞和分子水平,但整株水平上的研究也同样重要.整株水平上的钙吸收和运输包括根细胞的钙吸收、钙离子横向穿过根系并进入木质部、在木质部运输、从木质部移出并进入叶片或果实及在叶片或果实中运转分配等环节,既经过质外体也穿越共质体.钙离子通道、Ca2+-ATP酶和Ca2+/H+反向转运器等参与根细胞的钙吸收.在钙离子横向穿根进入木质部的过程中,需要穿越内皮层和木质部薄壁细胞组织.根系内皮层凯氏带阻挡了Ca2+沿质外体途径由内皮层外侧向内侧的移动,部分Ca2+由此通过离子通道流进内皮层细胞而转入共质体并到达木质部薄壁细胞组织,而由木质部薄壁细胞组织进入中柱质外体可能需要Ca2+-ATP酶驱动;还有一些Ca2+由内皮层细胞运出,沿内皮层内侧的质外体途径进入木质部导管,并通过导管运向枝干.钙离子以螯合态的形式在枝干导管运输;水流速率是影响钙离子沿导管运输的关键因子.钙离子在果实和叶片中的运输和分配不仅通过质外体途径也通过共质体途径.%Recently, research on Ca2+ transport in plants has been focused on cellular and molecular level.But the uptake, transport and distribution are also very important for calcium to accomplish its function at whole plant level. There are many cells along the way of transport of Ca2+ from root to shoot, and Ca2+ passes either through the cytoplasm of cells linked by plasmodesmata (the symplast) or through the spaces between cells (the apoplast), which include Ca2+ uptake by root cells, Ca2+ transport from root cortex to and through the xylem, and then out of it into leaves or fruits. Ca2+ channels, Ca2+/H+ antiporter and Ca2+-ATPase play roles in the uptake and transport of Ca2+ in root cells. To be transported from root surface to xylem,Ca2+ needs to traverse endodermal cells and

  18. Effects of dietary calcium and cadmium on cadmium accumulation, calcium and cadmium uptake from the water, and their interactions in juvenile rainbow trout

    Energy Technology Data Exchange (ETDEWEB)

    Baldisserotto, B. [Departamento de Fisiologia, Universidade Federal de Santa Maria, 97105.900 Santa Maria, RS (Brazil); Chowdhury, M.J. [Department of Biology, McMaster University, Hamilton, Ont., L8S 4K1 (Canada); Wood, Chris M. [Department of Biology, McMaster University, Hamilton, Ont., L8S 4K1 (Canada)]. E-mail: woodcm@mcmaster.ca

    2005-03-25

    The objective of this study was to examine the effects of chronically elevated dietary Ca{sup 2+} (as CaCO{sub 3}), alone and in combination with elevated dietary Cd, on survival, growth, and Cd and Ca{sup 2+} accumulation in several internal compartments in juvenile rainbow trout (Oncorhynchus mykiss). In addition, effects on short-term branchial uptake and internal distribution of newly accumulated waterborne Ca{sup 2+} and Cd during acute waterborne Cd exposure (50 {mu}g/L as CdNO{sub 3} for 3 h) were monitored using radiotracers ({sup 45}Ca, {sup 65}Cd). Fish were fed with four diets: 20 mg Ca{sup 2+}/g food (control), 50 mg Ca{sup 2+}/g food, 300 {mu}g Cd/g food, and 50 mg Ca{sup 2+}/g + 300 {mu}g Cd/g food for 30 days. There were no significant effects on growth, mortality, or total body Ca{sup 2+} accumulation. The presence of elevated Ca{sup 2+}, Cd, or Ca{sup 2+} + Cd in the diet all reduced waterborne Ca{sup 2+} uptake in a short-term experiment (3 h), though the inhibitory mechanisms appeared to differ. The effects were marked after 15 days of feeding, but attenuated by 30 days, except when the diet was elevated in both Ca{sup 2+} and Cd. The presence of elevated Ca{sup 2+} in the diet had only modest influence on Cd uptake from the water during acute Cd challenges but greatly depressed Cd uptake from the diet and accumulation in most internal tissues. None of the treatment diets prevented the decreases in waterborne Ca{sup 2+} uptake and new Ca{sup 2+} accumulation in internal tissues caused by acute exposure to waterborne Cd. In conclusion, there are complex interactions between waterborne and dietary effects of Ca{sup 2+} and Cd. Elevated dietary Ca{sup 2+} protects against both dietary and waterborne Cd uptake, whereas both waterborne and dietary Cd elevations cause reduced waterborne Ca{sup 2+} uptake.

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

    Science.gov (United States)

    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.

  20. Reduced myocardial {sup 18}F-FDG uptake after calcium channel blocker administration. Initial observation for a potential new method to improve plaque detection

    Energy Technology Data Exchange (ETDEWEB)

    Gaeta, Chiara; Flotats, Albert; Artigas, Carles; Deportos, Jordi; Geraldo, Llanos; Carrio, Ignasi [Sant Pau Hospital, PET Unit, Department of Nuclear Medicine, Barcelona (Spain); Fernandez, Yolanda [Center for Experimental Molecular Imaging (CIME), CETIR-ERESA, Barcelona (Spain); Pavia, Javier [Center for Experimental Molecular Imaging (CIME), CETIR-ERESA, Barcelona (Spain); Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona (Spain)

    2011-11-15

    Physiological glucose uptake by the myocardium may hamper visualization of coronary atherosclerotic plaques in {sup 18}F-FDG PET studies. Intracellular myocardial calcium relates to glucose influx. We assessed whether administration of a calcium channel blocker such as verapamil could decrease myocardial {sup 18}F-FDG uptake in mice. Experiments were conducted on ten male C57BL/6JOlaHsd mice. The mice were studied by {sup 18}F-FDG PET/CT under basal conditions and after a single administration of verapamil injected 1 h prior to {sup 18}F-FDG administration at doses of 1 mg/kg (group A, n = 5) and 20 mg/kg (group B, n = 5). PET scanning was started 60 min after injection of {sup 18}F-FDG employing a dedicated small-animal PET/CT system (ARGUS-CT). In each mouse, post-verapamil PET images were coregistered with the basal PET images. Volumetric regions of interest (VOI) were drawn on the basal study containing the myocardium of the whole left ventricle and quantitatively compared with the same VOI applied to the post-verapamil scan. The SUV{sub mean} was used to express the mean myocardial {sup 18}F-FDG uptake. The relative coefficient of variation (RV) between the basal and post-verapamil conditions was calculated. Verapamil administration decreased myocardial {sup 18}F-FDG uptake in all animals. The median (range) SUV{sub mean} values in group A were 2.6 (1.6-4.1) under basal conditions and 1.7 (1.1-2.9) after verapamil administration (p = 0.043), and in group B were 1.6 (1.3-2.0) under basal conditions and 1.0 (0.9-1.4) after verapamil administration (p = 0.043). The median (range) RV values were -31% (-5%, -50%) in group A, and -37% (-10%, -51%) in group B (p = 0.6). In this animal model there was a significant reduction in {sup 18}F-FDG uptake in the myocardium following verapamil administration. This type of intervention could facilitate the definition of coronary atherosclerotic plaque inflammation on {sup 18}F-FDG PET scans. (orig.)

  1. Uptake of CrO{sub 4}{sup 2-} ions by Fe-treated tri-calcium phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Serrano G, J.; Ramirez S, J. L.; Bonifacio M, J.; Granados C, F.; Badillo A, V. E., E-mail: juan.serrano@inin.gob.m [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2010-07-01

    CrO{sub 4}{sup 2-} ion adsorption of Fe-treated tri-calcium phosphate was studied by batch experiments as a function of contact time, initial concentration of metal ion and temperature. Adsorption results showed that at ph 5.5 and 1.0 x 10{sup -4} M chromium concentration the adsorption capacity of Fe-treated tri-calcium phosphate for CrO{sub 4}{sup 2-} ions was 7.10 x 10{sup -3} mmol/g. Chromium adsorption data on Fe-treated tri-calcium phosphate at various initial concentration fitted the Freundlich isotherm. By temperature studies the thermodynamic parameters {Delta}H{sup 0}, {Delta}G{sup 0} and {Delta}S{sup 0} were estimated and the obtained results showed that the adsorption reaction was endothermic and spontaneous. (Author)

  2. Regulation of the High-Affinity Nitrate Transport System in Wheat Roots by Exogenous Abscisic Acid and Glutamine

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Xue-Qiang Zhao; Yong-Guan Zhu; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2007-01-01

    Nitrate is a major nitrogen (N) source for most crops.Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels.Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency.The present study investigated the regulation of the high-affinity nitrate transport system (HATS) by exogenous abscisic acid (ABA) and glutamine (Gin) in wheat (Triticum aestivum L.) roots.Wheat seedlings grown in nutrient solution containing 2 mmollL nitrate as the only nitrogen source for 2 weeks were deprived of N for 4d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gin in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h.Treated wheat plants were then divided into two groups.One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L 15 N-labeled nitrate.The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate.Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction.When Gin was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced.These results imply that the inhibition by Gin of NRT2 expression occurs only when nitrate is present in the growth media.Although exogenous ABA and Gin induced HATS genes in the roots of wheat, they did not induce nitrate influx.

  3. In vivo effect of 3,5,3 prime -triiodothyronine on calcium uptake in several tissues in the rat: Evidence for a physiological role for calcium as the first messenger for the prompt action of thyroid hormone at the level of the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Segal, J. (Hebrew Univ. Hadassah Medical School, Jerusalem (Israel))

    1990-07-01

    Calcium has been shown in vitro to serve as the first messenger for the rapid effect of thyroid hormone at the level of the plasma membrane. In the present study the physiological relevance of this mechanism is examined in the whole animal. To this end, the effect of T3 on 45calcium uptake and sugar 2-deoxyglucose (2-DG) uptake, an effect that requires extracellular calcium, and the influence of calcium blockers thereon were measured in ventricles, atria, diaphragm, fat, and liver in the rat. In the first three tissues, T3 produced comparable changes in 45Ca uptake and 2-DG uptake (T3 increased 2-DG uptake in fat, where 45Ca uptake was undetected, and had no effect in liver); this activity was blocked by the calcium channel blocker cadmium. The effect of T3 on 45Ca uptake, like its effect on the in vivo uptake of 2-DG described previously, was biphasic and time related; at physiological doses of 0.01 and 0.1 micrograms/100 g BW, T3 increased 45Ca uptake, whereas at greater (pharmacological) doses of 1 and 100 micrograms/100 g BW, T3 was without effect or inhibited 45Ca uptake. In ventricles and atria, the stimulatory effect of T3 on 45Ca uptake was very rapid within 2 min, at which time it was at or near maximum (50-90% above control) and then declined gradually and was not seen after 10-20 min. Of the several calcium blockers employed, verapamil (organic) and cadmium (inorganic) were found to be the most effective. Verapamil and cadmium produced a rapid, transient, and dose-related inhibition of 45Ca uptake in the tissues examined (except fat tissue where, under the experimental conditions employed, 45Ca uptake was undetected). Verapamil, given iv (200 micrograms/100 g BW) or ip (1 mg/100 g BW), reduced tissue 45Ca uptake by 50-90% within 2 or 10 min, respectively, and then its inhibitory effect diminished rapidly and was not seen after 20-30 min.

  4. Monomeric TonB and the Ton box are required for the Formation of a High-Affinity Transporter-TonB Complex†

    Science.gov (United States)

    Freed, Daniel M.; Lukasik, Stephen M.; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S.

    2013-01-01

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In the present study, a soluble construct of Escherichia coli TonB (residues 33–239) was used to determine the affinity of TonB to the outer membrane transporters BtuB, FecA and FhuA. Using fluorescence anisotropy, TonB(33–239) was found to bind with high-affinity (tens of nM) to both BtuB and FhuA; however, no high-affinity binding was observed to FecA. In BtuB, the high affinity binding of TonB(33–239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33–239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33–239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When bound to the outer membrane transporter, DEER shows that the TonB(33–239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle. PMID:23517233

  5. Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

    Science.gov (United States)

    Freed, Daniel M; Lukasik, Stephen M; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S

    2013-04-16

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.

  6. Effects of ambient cadmium with calcium on mRNA expressions of calcium uptake related transporters in zebrafish (Danio rerio) larvae.

    Science.gov (United States)

    Liu, Chih-Tsen; Chou, Ming-Yi; Lin, Chia-Hao; Wu, Su Mei

    2012-08-01

    The mRNA expression levels of Ca²⁺ transporter genes including the epithelial calcium channel (ECaC), sodium/calcium exchanger 1b (NCX1b), and plasma membrane calcium ATPase 2 (PMCA2) were measured in zebrafish larvae after exposure to 0.08 μM Cd²⁺ in either water mixed with 0.2 mM Ca²⁺ (lCa) or 2 mM Ca²⁺ (hCa). The ECaC and NCX1b expression decreased at the 48 and 72 h mark, respectively; however, PMCA2 transcripts decreased at 96 h after exposure to Cd²⁺ in lCa environment. On the other hand, the ECaC transcripts were not affected; however, the PMCA2 transcripts were increased at 72 h, while the NCX1b transcripts significantly decreased at 48 and 96 h after exposure to Cd²⁺ in a hCa environment. The Ca²⁺ contents of larvae significantly decreased after Cd²⁺ exposure in a lCa environment; however, the Ca²⁺ contents were evidently higher after exposure to Cd²⁺ in a hCa environment, except for 48th h mark. In addition, ECaC morphants showed lower Ca²⁺ contents of whole-body, and there were higher levels of mortality after exposure to the same condition compared to the wild-type groups. In contrast, injection of ECaC cRNA resulted in an increase in Ca²⁺ content and the rate of Ca²⁺ influx in zebrafish embryos. Summary, the results showed that the Ca²⁺ transporters of zebrafish larvae were impacted after exposures of 0.08 μM Cd. However, in the exposure condition, the ECaC and PMCA2 transcripts could be restored to control levels after the fish were treated in an environment with hCa.

  7. High-affinity olfactory receptor for the death-associated odor cadaverine

    OpenAIRE

    2013-01-01

    Cadaverine and putrescine, two diamines emanating from decaying flesh, are strongly repulsive odors to humans but serve as innate attractive or social cues in other species. Here we show that zebrafish, a vertebrate model system, exhibit powerful and innate avoidance behavior to both diamines, and identify a high-affinity olfactory receptor for cadaverine.

  8. Isolation and cloning of the gene encoding high affinity phosphate transporter in rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    High affinity phosphate transporter plays an important role in plant adapting to low phosphorus. Isolation of genes coding this kind of protein has attracted worldwide scholars to accomplish. We aimed to isolate the gene and transfer it to target plants for breeding.

  9. Supramolecular surface immobilization of knottin derivatives for dynamic display of high affinity binders

    NARCIS (Netherlands)

    Sankaran, S.; Ruiter, de M.V.; Cornelissen, J.J.L.M.; Jonkheijm, P.

    2015-01-01

    Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly

  10. GHB receptor targets in the CNS: Focus on high-affinity binding sites

    DEFF Research Database (Denmark)

    Bay, Tina; Eghorn, Laura Friis; Klein, Anders Bue;

    2014-01-01

    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...

  11. N-Oxide analogs of WAY-100635 : new high affinity 5-HT (1A) receptor antagonists

    NARCIS (Netherlands)

    Oberwinkler - Marchais, Sandrine; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, H V

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective radiol

  12. N-Oxide analogs of WAY-100635 : new high affinity 5-HT1A receptor antagonists

    NARCIS (Netherlands)

    Marchais-Oberwinkler, S; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, HV

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective radiol

  13. Supramolecular surface immobilization of knottin derivatives for dynamic display of high affinity binders

    NARCIS (Netherlands)

    Sankaran, S.; de Ruiter, Mark Vincent; Cornelissen, Jeroen Johannes Lambertus Maria; Jonkheijm, Pascal

    2015-01-01

    Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly

  14. High affinity, bioavailable 3-amino-1,4-benzodiazepine-based gamma-secretase inhibitors.

    Science.gov (United States)

    Owens, Andrew P; Nadin, Alan; Talbot, Adam C; Clarke, Earl E; Harrison, Timothy; Lewis, Huw D; Reilly, Michael; Wrigley, Jonathan D J; Castro, José L

    2003-11-17

    In this paper, we describe the development of a novel series of high affinity, orally bioavailable 3-amino-1,4 benzodiazepine-based gamma-secretase inhibitors for the potential treatment of Alzheimer's disease. We disclose structure-activity relationships based around the 1, 3 and 5 positions of the benzodiazepine core structure.

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

    Science.gov (United States)

    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...

  16. The dual aptamer approach: rational design of a high-affinity FAD aptamer.

    Science.gov (United States)

    Merkle, T; Holder, I T; Hartig, J S

    2016-01-14

    A design strategy for high-affinity aptamers of complex biomolecules is presented. We developed an RNA with FAD-binding properties by combining known ATP- and FMN-aptamers. Cooperative binding of FAD was shown by SPR spectroscopy and fluorescence assays. The strategy should be transferable to several other biomolecules.

  17. Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Gidlöf, Ritha; Nielsen, Elsebet Østergaard

    2011-01-01

    Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K...

  18. Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Topp, M R; Blasie, J K

    1983-11-08

    The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.

  19. Use of low-calcium cultivars to reduce cadmium uptake and accumulation in edible amaranth (Amaranthus mangostanus L.).

    Science.gov (United States)

    He, Bao-Yan; Yu, Dan-Ping; Chen, Yan; Shi, Jia-Li; Xia, Yan; Li, Qu-Sheng; Wang, Li-Li; Ling, Ling; Zeng, Eddy Y

    2017-03-01

    This study aimed to investigate the mechanism of low Cd accumulation in crops using edible amaranth (Amaranthus mangostanus L.) as a model. Fifteen amaranth cultivars were grown in long-term contaminated soil, and the differences in soil Cd mobilization, root uptake, and root-shoot translocation between low- and high-Cd accumulating cultivars were examined. The transport pathways of Cd across the root were further identified in Hoagland nutrient solution using the Ca channel blocker La(3+), the ATP inhibitor 2, 4-dinitrophenol (DNP), and a nutrition-deficient culture. Cd concentrations in amaranth cultivars varied about six-fold and showed an elevated trend as the concentration of Ca and Zn increased (p low-Cd cultivars were significantly lower than those of high-Cd cultivars, and decreased with decreasing levels of soluble rhizosphere exudates. These findings indicated that low co-mobilization of Cd with essential metals mediated by root-induced exudates of low-Cd cultivars contributed to its low accumulation in amaranth. Uptake of Cd was inhibited along with Ca by La(3+) and DNP, but was promoted by Ca or Fe deficiency treatment. Therefore, the Ca pathway is likely the mode of Cd entry into amaranth roots, although Zn and Fe transporters may also be involved. Low-Ca cultivars exhibited lower Cd uptake capability than high-Ca cultivars. The low translocation efficiency of Cd from root to shoot also contributed to its low content accumulation in edible parts of amaranth.

  20. Calcium Mitigates Arsenic Toxicity in Rice Seedlings by Reducing Arsenic Uptake and Modulating the Antioxidant Defense and Glyoxalase Systems and Stress Markers.

    Science.gov (United States)

    Rahman, Anisur; Mostofa, Mohammad Golam; Alam, Md Mahabub; Nahar, Kamrun; Hasanuzzaman, Mirza; Fujita, Masayuki

    2015-01-01

    The effect of exogenous calcium (Ca) on hydroponically grown rice seedlings was studied under arsenic (As) stress by investigating the antioxidant and glyoxalase systems. Fourteen-day-old rice (Oryza sativa L. cv. BRRI dhan29) seedlings were exposed to 0.5 and 1 mM Na2HAsO4 alone and in combination with 10 mM CaCl2 (Ca) for 5 days. Both levels of As caused growth inhibition, chlorosis, reduced leaf RWC, and increased As accumulation in the rice seedlings. Both doses of As in growth medium induced oxidative stress through overproduction of reactive oxygen species (ROS) by disrupting the antioxidant defense and glyoxalase systems. Exogenous application of Ca along with both levels of As significantly decreased As accumulation and restored plant growth and water loss. Calcium supplementation in the As-exposed rice seedlings reduced ROS production, increased ascorbate (AsA) content, and increased the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), and the glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes compared with seedlings exposed to As only. These results suggest that Ca supplementation improves rice seedlings tolerance to As-induced oxidative stress by reducing As uptake, enhancing their antioxidant defense and glyoxalase systems, and also improving growth and physiological condition.

  1. Red blood cells of sickle cell disease patients exhibit abnormally high abundance of N-methyl D-aspartate receptors mediating excessive calcium uptake.

    Science.gov (United States)

    Hänggi, Pascal; Makhro, Asya; Gassmann, Max; Schmugge, Markus; Goede, Jeroen S; Speer, Oliver; Bogdanova, Anna

    2014-10-01

    Recently we showed that N-methyl D-aspartate receptors (NMDARs) are expressed in erythroid precursors (EPCs) and present in the circulating red blood cells (RBCs) of healthy humans, regulating intracellular Ca(2+) in these cells. This study focuses on investigating the possible role of NMDARs in abnormally high Ca(2+) permeability in the RBCs of patients with sickle cell disease (SCD). Protein levels of the NMDAR subunits in the EPCs of SCD patients did not differ from those in EPCs of healthy humans. However, the number and activity of the NMDARs in circulating SCD-RBCs was substantially up-regulated, being particularly high during haemolytic crises. The number of active NMDARs correlated negatively with haematocrit and haemoglobin levels in the blood of SCD patients. Calcium uptake via these non-selective cation channels was induced by RBC treatment with glycine, glutamate and homocysteine and was facilitated by de-oxygenation of SCD-RBCs. Oxidative stress and RBC dehydration followed receptor stimulation and Ca(2+) uptake. Inhibition of the NMDARs with an antagonist memantine caused re-hydration and largely prevented hypoxia-induced sickling. The EPCs of SCD patients showed higher tolerance to memantine than those of healthy subjects. Consequently, NMDARs in the RBCs of SCD patients appear to be an attractive target for pharmacological intervention.

  2. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.;

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article...

  3. Respiratory dysfunction by AFG3L2 deficiency causes decreased mitochondrial calcium uptake via organellar network fragmentation

    Science.gov (United States)

    Maltecca, Francesca; De Stefani, Diego; Cassina, Laura; Consolato, Francesco; Wasilewski, Michal; Scorrano, Luca; Rizzuto, Rosario; Casari, Giorgio

    2012-01-01

    The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca2+ uptake capacity. This defect is neither a consequence of global alteration in cellular Ca2+ homeostasis nor of the reduced driving force for Ca2+ internalization within mitochondria, since cytosolic Ca2+ transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca2+ uptake speed in Afg3l2−/− cells, indicating the presence of functional Ca2+ uptake machinery. Our results show that the defective Ca2+ handling in Afg3l2−/− cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca2+ elevations, hampering the proper Ca2+ diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2−/− MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca2+ buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca2+ homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations. PMID:22678058

  4. Role of calcium in the constriction of isolated cerebral arteries

    Energy Technology Data Exchange (ETDEWEB)

    Wendling, W.W.

    1987-01-01

    Calcium entry blockers (CEB) have been used in the experimental treatment or prevention of many cerebrovascular disorders including stroke, post-ischemic hypoperfusion after cardiac arrest, cerebral vasospasm after subarachnoid hemorrhage, and migraine headache. However, the mechanism of action of these drugs on the cerebral circulation is poorly understood. This study examined the effects of calcium antagonists, Ca/sup 2 +/-deficient solutions, and vasocostrictors on cerebrovascular tone and /sup 45/Ca fluxes, to determine the role of calcium in cerebral arterial constriction. A Scatchard plot of /sup 45/Ca binding to BMCA showed that Ca/sup 2 +/ was bound at either low or high affinity binding sties. The four vasoconstrictors (potassium, serotonin, PGF/sub 2 ..cap alpha../, or SQ-26,655) each increased low affinity /sup 45/Ca uptake into BMCA. The results demonstrate that: (1) Potassium and serotonin constrict BMCA mainly by promoting Ca/sup 2 +/ influx through CEB-sensitive channels; (2) PGF/sub 2 ..cap alpha../ and SQ-26,655 constrict BMCA in part by promoting Ca/sup 2 +/ influx through CEB-sensitive channels, and in part by releasing Ca/sup 2 +/ from depletable internal stores; (3) The major action of CEB on BMCA is to block vasoconstrictor-induced Ca/sup 2 +/ uptake through both potential-operated (K/sup +/-stimulated) and receptor-operated channels.

  5. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    XIANG YongZhe; WANG Na; ZHANG Ji; LI Kun; ZHANG ZhongWei; LIN HongHui; YU XiaoQi

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction be-tween 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane.High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA con-densation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  6. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction between 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod-odecane. High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover,the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA condensation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

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

    Science.gov (United States)

    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.

  8. A High-Affinity Metal-Binding Peptide From Escherichia Coli Hypb

    Energy Technology Data Exchange (ETDEWEB)

    Chung, K.C.Chan; Cao, L.; Dias, A.V.; Pickering, I.J.; George, G.N.; Zamble, D.B.

    2009-05-12

    The high-affinity nickel-binding site of the Escherichia coli [NiFe]-hydrogenase accessory protein HypB was localized to residues at the immediate N-terminus of the protein. Modification of a metal-binding fusion protein, site-directed mutagenesis experiments, and DFT calculations were used to identify the N-terminal amine as a ligand as well as the three cysteine residues in the CXXCGCXXX motif. This sequence can be removed from the protein and both a synthesized peptide and a protein fusion bind nickel with a similar affinity and the same structure as the parent metalloprotein, indicating the self-sufficiency of this high-affinity nickel-binding sequence.

  9. Cubilin, a High Affinity Receptor for Fibroblast Growth Factor 8, Is Required for Cell Survival in the Developing Vertebrate Head*

    Science.gov (United States)

    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

  10. Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

    Science.gov (United States)

    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.

  11. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.

    Science.gov (United States)

    Maute, Roy L; Gordon, Sydney R; Mayer, Aaron T; McCracken, Melissa N; Natarajan, Arutselvan; Ring, Nan Guo; Kimura, Richard; Tsai, Jonathan M; Manglik, Aashish; Kruse, Andrew C; Gambhir, Sanjiv S; Weissman, Irving L; Ring, Aaron M

    2015-11-24

    Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

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

    Science.gov (United States)

    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. Nuclear Choline Acetyltransferase Activates Transcription of a High-affinity Choline Transporter*

    OpenAIRE

    Matsuo, Akinori; Bellier, Jean-Pierre; Nishimura, Masaki; YASUHARA, Osamu; Saito, Naoaki; Kimura, Hiroshi

    2010-01-01

    Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

    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. Enhanced selection of high affinity DNA-reactive B cells following cyclophosphamide treatment in mice.

    Directory of Open Access Journals (Sweden)

    Daisuke Kawabata

    Full Text Available A major goal for the treatment of patients with systemic lupus erythematosus with cytotoxic therapies is the induction of long-term remission. There is, however, a paucity of information concerning the effects of these therapies on the reconstituting B cell repertoire. Since there is recent evidence suggesting that B cell lymphopenia might attenuate negative selection of autoreactive B cells, we elected to investigate the effects of cyclophosphamide on the selection of the re-emerging B cell repertoire in wild type mice and transgenic mice that express the H chain of an anti-DNA antibody. The reconstituting B cell repertoire in wild type mice contained an increased frequency of DNA-reactive B cells; in heavy chain transgenic mice, the reconstituting repertoire was characterized by an increased frequency of mature, high affinity DNA-reactive B cells and the mice expressed increased levels of serum anti-DNA antibodies. This coincided with a significant increase in serum levels of BAFF. Treatment of transgene-expressing mice with a BAFF blocking agent or with DNase to reduce exposure to autoantigen limited the expansion of high affinity DNA-reactive B cells during B cell reconstitution. These studies suggest that during B cell reconstitution, not only is negative selection of high affinity DNA-reactive B cells impaired by increased BAFF, but also that B cells escaping negative selection are positively selected by autoantigen. There are significant implications for therapy.

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

    Science.gov (United States)

    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.

  17. [3H]ATPA: a high affinity ligand for GluR5 kainate receptors.

    Science.gov (United States)

    Hoo, K; Legutko, B; Rizkalla, G; Deverill, M; Hawes, C R; Ellis, G J; Stensbol, T B; Krogsgaard-Larsen, P; Skolnick, P; Bleakman, D

    1999-12-01

    The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

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

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available 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.

  19. Structural Basis for High-Affinity Peptide Inhibition of Human Pin1

    Science.gov (United States)

    Zhang, Yan; Daum, Sebastian; Wildemann, Dirk; Zhou, Xiao Zhen; Verdecia, Mark A.; Bowman, Marianne E.; Lücke, Christian; Hunter, Tony; Lu, Kun-Ping; Fischer, Gunter; Noel, Joseph P.

    2009-01-01

    Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I β-turn conformation for Pin1 prolyl peptide isomerase domain–peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors. PMID:17518432

  20. Effects of calcium peroxide on arsenic uptake by celery (Apium graveolens L.) grown in arsenic contaminated soil.

    Science.gov (United States)

    Liu, Chuan-ping; Luo, Chun-ling; Xu, Xiang-hua; Wu, Chuang-an; Li, Fang-bai; Zhang, Gan

    2012-03-01

    The ability of calcium peroxide (CaO(2)) to immobilize As of contaminated soil was studied using pot and field experiments. In pot experiment, CaO(2) applied at 2.5 and 5 g kg(-1) significantly increased celery shoot weight and decreased shoot As accumulation, which was ascribed to the formation of stable crystalline Fe and Al oxides bound As and the reduction of labile As fractions in the soil. The labile As fractions were pH dependent and it followed a "V" shaped profile with the change of pH. In field experiment, the dose of CaO(2) application at 750 kg ha(-1) was optimal and at which the celery was found to produce the highest biomass (63.4 Mg ha(-1)) and lowest As concentration (0.43 mg kg(-1)). CaO(2) probably has a promising potential as soil amendment to treat As contaminated soils.

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

    Science.gov (United States)

    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

  2. Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

    Science.gov (United States)

    Erickson, Jeffrey D

    2017-07-01

    Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K(+) -stimulated MeAIB transport displays an affinity (Km ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca(2+) , and is blocked by inhibition of voltage-gated Ca(2+) channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca(2+) and voltage-gated calcium channels, but is also blocked by the Na(+) channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca(2+) , but on Na(+) ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K(+) -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017

  3. Investigating a potential mechanism of Cd resistance in Chironomus riparius larvae using kinetic analysis of calcium and cadmium uptake

    Energy Technology Data Exchange (ETDEWEB)

    Gillis, Patricia L. [Department of Biology, McMaster University, Hamilton, ON, L8S-4K1 (Canada)], E-mail: patty.gillis@ec.gc.ca; Wood, Chris M. [Department of Biology, McMaster University, Hamilton, ON, L8S-4K1 (Canada)

    2008-09-17

    The uptake kinetics of waterborne Ca and Cd, both independently and in combination, were examined in C. riparius larvae, which are extremely Cd tolerant. Larvae exposed to Ca (100-2500 {mu}mol L{sup -1}), exhibited classic Michaelis-Menten saturation kinetics for Ca influx, measured using {sup 45}Ca as a radio-tracer. The maximum rate of Ca influx (J{sub max}{sup Ca}) was 0.39 {mu}mol g{sup -1} h{sup -1}, and the Ca concentration where the carrier reached half saturation (K{sub M}{sup Ca}) was 289 {mu}mol L{sup -1}. Cd influx was measured using {sup 109}Cd as a radio-tracer in larvae exposed to Cd (0-1400 {mu}mol L{sup -1}) while the Ca concentration was set to the K{sub M}{sup Ca}. This revealed a J{sub max}{sup Cd} (2.26 {mu}mol g{sup -1} h{sup -1}) which was nearly 6-fold higher that of Ca. This unusually high capacity for Cd uptake is in accordance with the huge tissue Cd burdens that chironomid larvae are able to accumulate during high level exposures. The apparent K{sub M}{sup Cd} (1133 {mu}mol Cd L{sup -1}), when recalculated to account for the background Ca level, was still high (567 {mu}mol Cd L{sup -1}), suggesting that this organism has a low affinity for Cd relative to most aquatic animals, indeed lower or comparable to its affinity for Ca. In consequence, even well above environmentally relevant Cd exposures, C. riparius does not accumulate Cd at the expense of Ca, thereby avoiding internal hypocalcaemia, in contrast to most other organisms which are much more sensitive to Cd. However, Ca influx was significantly reduced when 1200 {mu}mol Cd L{sup -1} was added to Ca exposures (96-2410 {mu}mol L{sup -1}). Michaelis-Menten analysis revealed a similar J{sub max}{sup Ca} in Cd-exposed and control larvae (i.e. exposed only to Ca), but that the apparent K{sub M}{sup Ca} was many-fold higher in larvae which were simultaneously exposed to Ca and Cd. Conversely, increasing Ca concentrations (96-2410 {mu}mol L{sup -1}) progressively inhibited Cd uptake from a

  4. Effect of calcium carbonate on cadmium and nutrients uptake in tobacco (Nicotiana tabacum L.) planted on contaminated soil.

    Science.gov (United States)

    Zeng, Wei-Ai; Li, Fan; Zhou, Hang; Qin, Xiao-Li; Zou, Zi-Jin; Tian, Tao; Zeng, Min; Liao, Bo-Han

    2016-01-01

    In the present study, calcium carbonate (CaCO3) was applied to Cd-contaminated soil at rates of 0, 0.5 and 1.0 g kg(-1). The effect of CaCO3 on soil pH, organic matter, available Cd, exchangeable Cd and level of major nutrients in a tobacco field and on accumulation of various elements in tobacco plants was determined. The results showed that CaCO3 application significantly increased the pH level, available P and exchangeable Ca but decreased organic matter, available Cd, exchangeable Cd, available heavy metals (Fe, Mn, Zn and Cu) and available K in soil. Additionally, CaCO3 application substantially reduced Cd accumulation in tobacco roots, stems, upper leaves, middle leaves and lower leaves, with maximum decrease of 22.3%, 32.1%, 24.5%, 22.0% and 18.2%, respectively. There were large increase in total Ca and slight increases in total N and K but decrease to varying degrees in total Fe, Cu and Zn due to CaCO3 application. CaCO3 had little effect on total P and Mn levels in tobacco leaves.

  5. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by proteoliposomes and cultured rat sertoli cells: Evidence for involvement of voltage-activated and voltage-independent calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1989-12-01

    We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-(3-thiotriphosphate)) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels.

  6. Calcium- and ammonium ion-modification of zeolite amendments affects the metal-uptake of Hieracium piloselloides in a dose-dependent way.

    Science.gov (United States)

    Peter, Anca; Mihaly-Cozmuta, Leonard; Mihaly-Cozmuta, Anca; Nicula, Camelia; Indrea, Emil; Tutu, Hlanganani

    2012-10-26

    The role of natural zeolite and of two types of modified zeolite (with ammonium and with calcium ions) in reducing the accumulation of ions of heavy metals in roots and leaves of Hieracium piloselloides grown on tailing ponds was investigated. The variation of the content of zeolite (5% w/w and 10% w/w) is another parameter that significantly and positively affects the accumulation of the metal ions in Hieracium piloselloides. The results showed that zeolite used as an amendment in the soil in tailing ponds significantly reduces the accumulation of heavy metal ions in Hieracium piloselloides. The highest concentrations of heavy metals were accumulated in plants grown on soil without zeolite, being followed by the plants grown on the substrate with natural zeolite. Moreover, the translocation factors of heavy metal ions uptake in roots and leaves grown on substrates with modified zeolites are lower than those calculated for the organs grown on the substrate amended with natural zeolite. This behaviour has demonstrated the positive effect of those changes of zeolite amendments in the potential phytoremediation practice.

  7. Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (Fc epsilon RI)

    OpenAIRE

    1992-01-01

    It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast c...

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

    Energy Technology Data Exchange (ETDEWEB)

    Yliniemelae, A.; Gynther, J. (Univ. of Kuopio (Finland)); Konschin, H.; Tylli, H. (Univ. of Helsinki (Finland)); Rouvinen, J. (Univ. of Joensuu (Finland))

    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. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  10. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

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

    Science.gov (United States)

    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

  12. High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

    Institute of Scientific and Technical Information of China (English)

    Biplab Bose; Navin Khanna; Subrat K Acharya; Subrata Sinha

    2005-01-01

    AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS: We cloned the VH and VL genes of this mouse antibody; and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. Coli.RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal.This chimeric antibody fragment was further expressed in different strains of E> coli to increase the yield.CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.

  13. Genetic evidence of a high-affinity cyanuric acid transport system in Pseudomonas sp. ADP.

    Science.gov (United States)

    Platero, Ana I; Santero, Eduardo; Govantes, Fernando

    2014-03-01

    The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here, we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiting cyanuric acid concentrations is consistent with high-affinity transport of this substrate. Acquisition of the ability to grow at low cyanuric acid concentrations upon conjugal transfer of pADP1 to the nondegrading host Pseudomonas putida KT2442 suggests that all activities required for this phenotype are encoded in this plasmid. Co-expression of the pADP1-borne atzDEF and atzTUVW genes, encoding the cyanuric acid utilization pathway and the subunits of an ABC-type solute transport system, in P. putida KT2442 was sufficient to promote growth at cyanuric acid concentrations as low as 50 μM in batch culture. Taken together, our results strongly suggest that the atzTUVW gene products are involved in high-affinity transport of cyanuric acid.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  17. Role of the human high-affinity copper transporter in copper homeostasis regulation and cisplatin sensitivity in cancer chemotherapy.

    Science.gov (United States)

    Kuo, Macus Tien; Fu, Siqing; Savaraj, Niramol; Chen, Helen H W

    2012-09-15

    The high-affinity copper transporter (Ctr1; SCLC31A1) plays an important role in regulating copper homeostasis because copper is an essential micronutrient and copper deficiency is detrimental to many important cellular functions, but excess copper is toxic. Recent research has revealed that human copper homeostasis is tightly controlled by interregulatory circuitry involving copper, Sp1, and human (hCtr1). This circuitry uses Sp1 transcription factor as a copper sensor in modulating hCtr1 expression, which in turn controls cellular copper and Sp1 levels in a 3-way mutual regulatory loop. Posttranslational regulation of hCtr1 expression by copper stresses has also been described in the literature. Because hCtr1 can also transport platinum drugs, this finding underscores the important role of hCtr1 in platinum-drug sensitivity in cancer chemotherapy. Consistent with this notion is the finding that elevated hCtr1 expression was associated with favorable treatment outcomes in cisplatin-based cancer chemotherapy. Moreover, cultured cell studies showed that elevated hCtr1 expression can be induced by depleting cellular copper levels, resulting in enhanced cisplatin uptake and its cell-killing activity. A phase I clinical trial using a combination of trientine (a copper chelator) and carboplatin has been carried out with encouraging results. This review discusses new insights into the role of hCtr1 in regulating copper homeostasis and explains how modulating cellular copper availability could influence treatment efficacy in platinum-based cancer chemotherapy through hCtr1 regulation.

  18. Sequestration of Sr(II) by calcium oxalate—A batch uptake study and EXAFS analysis of model compounds and reaction products

    Science.gov (United States)

    Singer, David M.; Johnson, Stephen B.; Catalano, Jeffrey G.; Farges, François; Brown, Gordon E., Jr.

    2008-10-01

    Calcium oxalate monohydrate (CaC 2O 4·H 2O—abbreviated as CaOx) is produced by two-thirds of all plant families, comprising up to 80 wt.% of the plant tissue and found in many surface environments. It is unclear, however, how CaOx in plants and soils interacts with metal ions and possibly sequesters them. This study examines the speciation of Sr(II) aq following its reaction with CaOx. Batch uptake experiments were conducted over the pH range 4-10, with initial Sr solution concentrations, [Sr] aq, ranging from 1 × 10 -4 to 1 × 10 -3 M and ionic strengths ranging of 0.001-0.1 M, using NaCl as the background electrolyte. Experimental results indicate that Sr uptake is independent of pH and ionic strength over these ranges. After exposure of CaOx to Sr aq for two days, the solution Ca concentration, [Ca] aq, increased for all samples relative to the control CaOx suspension (with no Sr added). The amount of Sr aq removed from solution was nearly equal to the total [Ca] aq after exposure of CaOx to Sr. These results suggest that nearly 90% of the Sr is removed from solution to a solid phase as Ca is released into solution. We suggest that the other 10% is sequestered through surface adsorption on a solid phase, although we have no direct evidence for this. Extended X-ray absorption fine structure (EXAFS) spectroscopy was used to determine the molecular-level speciation of Sr in the reaction products. Deconvolutions of the Sr K-edge EXAFS spectra were performed to identify multi-electron excitation (MEE) features. MEE effects were found to give rise to low-frequency peaks in the Fourier transform before the first shell of oxygen atoms and do not affect EXAFS fitting results. Because of potential problems caused by asymmetric distributions of Sr-O distances when fitting Sr K-edge EXAFS data using the standard harmonic model, we also employed a cumulant expansion model and an asymmetric analytical model to account for anharmonic effects in the EXAFS data. For Sr

  19. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    OpenAIRE

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H. F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affin...

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

    Energy Technology Data Exchange (ETDEWEB)

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. (Univ. of Maryland, Baltimore (USA))

    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.

  1. The Glucose Sensor-Like Protein Hxs1 Is a High-Affinity Glucose Transporter and Required for Virulence in Cryptococcus neoformans

    Science.gov (United States)

    Baker, Gregory M.; Fahmy, Hany; Jiang, Linghuo; Xue, Chaoyang

    2013-01-01

    Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence. PMID:23691177

  2. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death.

    Science.gov (United States)

    Norholm, Morten H H; Nour-Eldin, Hussam H; Brodersen, Peter; Mundy, John; Halkier, Barbara A

    2006-04-17

    We report the biochemical characterization in Xenopus oocytes of the Arabidopsis thaliana membrane protein, STP13, as a high affinity, hexose-specific H(+)-symporter. Studies with kinase activators suggest that it is negatively regulated by phosphorylation. STP13 promoter GFP reporter lines show GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants.

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

    Science.gov (United States)

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

    2016-08-31

    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.

  4. Selection of high affine peptide ligands for detection of Clostridium Tyrobutyricum spores.

    Science.gov (United States)

    Lavilla, María; De Luis, Ruth; Pérez, María Dolores; Calvo, Miguel; Sánchez, Lourdes

    2009-11-01

    Clostridium tyrobutyricum is the main agent responsible for "late blowing" in cheese, which causes severe economic losses. Nowadays, the reference method for its detection is the Most-Probable-Number (MPN); however, it is time consuming and non-specific. Thus, in order to check milk contamination with spores of C. tyrobutyricum, a more specific and rapid method would be required. The objective of this work was to obtain a ligand to establish the basis to develop a biomagnetic separation method for detection of C. tyrobutyricum spores. This study describes the selection of thirteen highly affine peptides to C. tyrobutyricum spores from a phage-display peptide library. In order to test the ability of the peptides attached to a solid support to bind the spores, the most frequent peptide was synthesised and used to coat paramagnetic beads.

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

    Science.gov (United States)

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

    2013-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Sarah E. Altschuler

    2013-09-01

    Full Text Available 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, Schizosaccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

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

    Science.gov (United States)

    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.

  8. Cytisine derivatives as high affinity nAChR ligands: synthesis and comparative molecular field analysis.

    Science.gov (United States)

    Nicolotti, O; Canu Boido, C; Sparatore, F; Carotti, A

    2002-06-01

    A number of new N-substituted cytisine derivatives were prepared and tested, along with similar compounds already described by us and others, as high affinity neuronal acetylcholine receptor ligands. Structure-affinity relationships were discussed in the light of our recently proposed pharmacophore model for nicotinic receptor agonists. The most significant physicochemical interactions modulating the receptor-ligand binding were detected at the three dimensional (3D) level by means of comparative molecular field analysis (CoMFA). The best predictive PLS model was a single-field steric model showing good statistical figures: n = 17, Q2 = 0.717, s(ev) = 0.566, r2 = 0.942, s = 0.275.

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

    Science.gov (United States)

    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.

  10. Structural insights into a high affinity nanobody:antigen complex by homology modelling

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2017-01-01

    B binding were identified and used as input to the docking. Furthermore, residues likely involved in the RgpB epitope was identified based upon RgpB:RgpA alignment and analysis of residue surface accessibility. CDR residues and putitative RgpB epitope residues were used as input to an information-driven...... flexible docking approach using the HADDOCK server. Analysis of the VHH7:RgpB model demonstrated that the epitope was found in the immunoglobulin-like domain and residue pairs located at the molecular paratope:epitope interface important for complex stability was identified. Collectively, the VHH7 homology...... model and VHH7:RgpB docking supplies knowledge of the residues involved in the high affinity interaction. This information could prove valuable in the design of an antibody-drug conjugate for specific RgpB targeting....

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

    Directory of Open Access Journals (Sweden)

    S.F. Marino

    2016-03-01

    Full Text Available 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.

  12. Expression of a Hybrid Human Superoxide Dismutase with a High Affinity for Heparin

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A designed heparin-affinity of human Cu, Zn-SOD is described. The natural leader peptide of P.leiognathi Cu, Zn-SOD and a heparin-binding peptide containing a stretch of 7 Arg were fused to the N-terminal and the C-terminal of human Cu, Zn-SOD respectively. The resulted hybrid enzyme had not only a normal SOD activity but also a high affinity for heparin eluted on the heparin-Sepharose column at 0.4 mol/L NaCl. Some properties, such as the optimum pH, the thermostability and the half-life in the circulation of rats, were also analyzed.

  13. Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor

    DEFF Research Database (Denmark)

    Rao, C N; Castronovo, V; Schmitt, M C;

    1989-01-01

    The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et...... al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7137-7141]. Primer extension experiments demonstrated that the clone contained the complete 5' sequence of the murine laminin receptor mRNA. RNA blot data demonstrated a single-sized laminin receptor mRNA, approximately 1400 bases long, in human, mouse......, and rat. The nascent laminin receptor predicted from the cDNA sequence is 295 amino acids long, with a molecular weight of 33,000, and contains one intradisulfide bridge, a short putative transmembrane domain, and an extracellular carboxy-terminal region which has abundant glutamic acid residues...

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    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.

  16. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  17. Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging.

    Science.gov (United States)

    Dhar, Alok; Feiss, Michael

    2005-03-18

    DNA packaging by large DNA viruses such as the tailed bacteriophages and the herpesviruses involves DNA translocation into a preformed protein shell, called the prohead. Translocation is driven by an ATP hydrolysis-powered DNA packaging motor. The bacteriophages encode a heterodimeric viral DNA packaging protein, called terminase. The terminases have an ATPase center located in the N terminus of the large subunit implicated in DNA translocation. In previous work with phage lambda, lethal mutations that changed ATP-reactive residues 46 and 84 of gpA, the large terminase subunit, were studied. These mutant enzymes retained the terminase endonuclease and helicase activities, but had severe defects in virion assembly, and lacked the terminase high-affinity ATPase activity. Surprisingly, in the work described here, we found that enzymes with the conservative gpA changes Y46F and Y46A had only mild packaging defects. These mild defects contrast with their profound virion assembly defects. Thus, these mutant enzymes have, in addition to the mild DNA packaging defects, a severe post-DNA packaging defect. In contrast, the gpA K84A enzyme had similar virion assembly and DNA packaging defects. The DNA packaging energy budget, i.e. DNA packaged/ATP hydrolyzed, was unchanged for the mutant enzymes, indicating that DNA translocation is tightly coupled to ATP hydrolysis. A model is proposed in which gpA residues 46 and 84 are important for terminase's high-affinity ATPase activity. Assembly of the translocation complex remodels this ATPase so that residues 46 and 84 are not crucial for the activated translocation ATPase. Changing gpA residues 46 and 84 primarily affects assembly, rather than the activity, of the translocation complex.

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

    Science.gov (United States)

    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.

  19. Measuring the serotonin uptake site using (/sup 3/H)paroxetine--a new serotonin uptake inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Gleiter, C.H.; Nutt, D.J.

    1988-01-01

    Serotonin is an important neurotransmitter that may be involved in ethanol preference and dependence. It is possible to label the serotonin uptake site in brain using the tricyclic antidepressant imipramine, but this also binds to other sites. We have used the new high-affinity uptake blocker paroxetine to define binding to this site and report it to have advantages over imipramine as a ligand.

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

    Science.gov (United States)

    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.

  1. Design and Investigation of a [(18)F]-Labeled Benzamide Derivative as a High Affinity Dual Sigma Receptor Subtype Radioligand for Prostate Tumor Imaging.

    Science.gov (United States)

    Yang, Dongzhi; Comeau, Anthony; Bowen, Wayne D; Mach, Robert H; Ross, Brian D; Hong, Hao; Van Dort, Marcian E

    2017-03-06

    High overexpression of sigma (σ) receptors (σ1 and σ2 subtypes) in a variety of human solid tumors has prompted the development of σ receptor-targeting radioligands, as imaging agents for tumor detection. A majority of these radioligands to date target the σ2 receptor, a potential marker of tumor proliferative status. The identification of approximately equal proportions of both σ receptor subtypes in prostate tumors suggests that a high affinity, dual σ receptor-targeting radioligand could potentially provide enhanced tumor targeting efficacy in prostate cancer. To accomplish this goal, we designed a series of ligands which bind to both σ receptor subtypes with high affinity. Ligand 3a in this series, displaying optimal dual σ receptor subtype affinity (σ1, 6.3 nM; σ2, 10.2 nM) was radiolabeled with fluorine-18 ((18)F) to give [(18)F]3a and evaluated as a σ receptor-targeting radioligand in the mouse PC-3 prostate tumor model. Cellular assays with PC-3 cells demonstrated that a major proportion of [(18)F]3a was localized to cell surface σ receptors, while ∼10% of [(18)F]3a was internalized within cells after incubation for 3.5 h. Serial PET imaging in mice bearing PC-3 tumors revealed that uptake of [(18)F]3a was 1.6 ± 0.8, 4.4 ± 0.3, and 3.6 ± 0.6% ID/g (% injection dose per gram) in σ receptor-positive prostate tumors at 15 min, 1.5 h, and 3.5 h postinjection, respectively (n = 3) resulting in clear tumor visualization. Blocking studies conducted with haloperidol (a nonselective inhibitor for both σ receptor subtypes) confirmed that the uptake of [(18)F]3a was σ receptor-mediated. Histology analysis confirmed similar expression of σ1 and σ2 in PC-3 tumors which was significantly greater than its expression in normal organs/tissues such as liver, kidney, and muscle. Metabolite studies revealed that >50% of radioactivity in PC-3 tumors at 30 min postinjection represented intact [(18)F]3a. Prominent σ receptor-specific uptake of [(18)F]3a in

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Aili Bao

    2015-04-01

    Full Text Available 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.

  4. Time-resolved investigation of molecular components involved in the induction of NO3- high affinity transport system in maize roots

    Directory of Open Access Journals (Sweden)

    Youry Pii

    2016-11-01

    Full Text Available 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 behaviour 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 nondenaturing 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.

  5. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    Science.gov (United States)

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  6. New synthesis and tritium labeling of a selective ligand for studying high-affinity γ-hydroxybutyrate (GHB) binding sites.

    Science.gov (United States)

    Vogensen, Stine B; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H F; Clausen, Rasmus P

    2013-10-24

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [(3)H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity, and we demonstrate in vivo brain penetration. In vitro characterization of [(3)H]-1 binding shows high specificity to the high-affinity GHB binding sites.

  7. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-Affinity γ-Hydroxybutyrate (GHB) Binding Sites

    DEFF Research Database (Denmark)

    Vogensen, Stine B.; Marek, Ales; Bay, Tina

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide....... Screening of 1 against different CNS targets establishes a high selectivity, and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites....

  8. Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

    Science.gov (United States)

    Reis, C R; van der Sloot, A M; Natoni, A; Szegezdi, E; Setroikromo, R; Meijer, M; Sjollema, K; Stricher, F; Cool, R H; Samali, A; Serrano, L; Quax, W J

    2010-10-21

    The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

  9. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    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. PMID:23747010

  10. Early signs of pathological cognitive aging in mice lacking high-affinity nicotinic receptors.

    Directory of Open Access Journals (Sweden)

    Eleni eKonsolaki

    2016-04-01

    Full Text Available 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. Α 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 behavioural 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 behaviours, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm and extend the hypothesis that β2-/- animals exhibit age-related cognitive impairments, manifested in both spatial learning and recognition memory tasks. In addition, we reveal deficits in spontaneous behaviour and habituation processes earlier in life. To our knowledge, this is the first study to perform an extensive behavioural examination of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioural changes to global dementia due to the combined effect of the neuropathology and aging.

  11. Glycation of the high affinity NGF-receptor and RAGE leads to reduced ligand affinity.

    Science.gov (United States)

    Bennmann, Dorit; Kannicht, Christoph; Fisseau, Claudine; Jacobs, Kathleen; Navarette-Santos, Alexander; Hofmann, Britt; Horstkorte, Rüdiger

    2015-09-01

    AGEs are posttranslational modifications generated by irreversible non-enzymatic crosslinking reactions between sugars and proteins - a reaction referred to as glycation. Glycation, a feature of ageing, can lead to non-degradable and less functional proteins and enzymes and can additionally induce inflammation and further pathophysiological processes such as neurodegeneration. In this study we investigated the influence of glycation on the high affinity NGF-receptor TrkA and the AGE-receptor RAGE. We quantified the binding affinity of the TrkA-receptor and RAGE to their ligands by surface plasmon resonance (SPR) and compared these to the binding affinity after glycation. At the same time, we established a glycation procedure using SPR. We found that glycation of TrkA reduced the affinity to NGF by a factor of three, which could be shown to lead to a reduction of NGF-dependent neurite outgrowth in PC12 cells. Glycation of RAGE reduced binding affinity of AGEs by 10-fold.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

    Directory of Open Access Journals (Sweden)

    Kaval Kaur

    Full Text Available Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE. Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

  15. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

    Science.gov (United States)

    Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T.; Henry Dunand, Carole J.; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L.; Munroe, Melissa E.; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A.; Wilson, Patrick C.

    2015-01-01

    Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus. PMID:25951191

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

    Directory of Open Access Journals (Sweden)

    Amanda E Siglin

    Full Text Available Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC and the dynactin p150(Glued; however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued. Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued. Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

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

    Science.gov (United States)

    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

  1. [The high-affinity IgE receptor: lessons from structural analysis].

    Science.gov (United States)

    Blank, Ulrich; Jouvin, Marie-Hélène; Guérin-Marchand, Claudine; Kinet, Jean-Pierre

    2003-01-01

    The high affinity receptor for IgE, FcERI, is at the core of the allergic reaction. This receptor is expressed mainly on mast cells and basophils. Interaction of an allergen with its specific IgE bound to FcERI triggers cell activation, which induces the release of numerous mediators that are responsible for allergic manifestations. The recent increase in the prevalence of allergic diseases in developed countries has resulted in renewed efforts towards the development of new drugs. One of these is a humanised antibody directed against the IgE ligand. This antibody recognises specifically free but not FcERI-bound IgE thus preventing ligand binding and subsequent cell activation. This antibody has shown some efficacy in clinical trials involving patients with asthma and allergic rhinitis. The recent elucidation of the tridimensional structure of the complex between IgE and FcERI provides unexpected information regarding the mechanism of assembly of the complex, which now can be used to design small chemical compounds capable of specifically inhibiting this interaction.

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

    Energy Technology Data Exchange (ETDEWEB)

    Raza, H.; Chung, W.L.; Mukhtar, H. (Department of Dermatology, University Hospitals of Cleveland, Case Western Reserve University, OH (USA))

    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.

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

    Science.gov (United States)

    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.

  4. Sequential administration of the high affinity CXCR4 antagonist BKT140 promotes megakaryopoiesis and platelet production.

    Science.gov (United States)

    Abraham, Michal; Weiss, Ido D; Wald, Hanna; Wald, Ori; Nagler, Arnon; Beider, Katia; Eizenberg, Orly; Peled, Amnon

    2013-10-01

    Platelets are the terminal differentiation product of megakaryocytes (MKs). Cytokines, such as thrombopoietin (TPO), are known to influence different steps in MK development; however, the complex differentiation and platelet localization processes are not fully understood. MKs express the receptor CXCR4 and have been shown to migrate in response to CXCL12 and to increase their platelet production. In this study, we studied the role of CXCR4 in platelet production with the high affinity CXCR4 antagonist, BKT140. Single and sequential administration of BKT140 significantly increased the number of MKs and haematopoietic progenitors (HPCs) within the bone marrow (BM). Increased megakaryopoiesis was associated with increased platelet production. Single and sequential administration of BKT140 also increased the number of HPCs in the blood. In a model of 5-fluorouracil-induced thrombocytopenia, BKT140 significantly reduced the severity and duration of thrombocytopenia and cytopenia when administered before and after chemotherapy. Our results demonstrated that the CXCR4 antagonist, BKT140, mediated unique beneficial effects by stimulating megakaryopoiesis and platelet production. These results provide evidence for the possible therapeutic use of BKT140 for modulating platelet numbers in thrombocytopenic conditions. © 2013 John Wiley & Sons Ltd.

  5. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  6. A high-affinity, radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine.

    Science.gov (United States)

    Bray, Lauriane; Moulédous, Lionel; Tafani, Jean A M; Germanier, Maryse; Zajac, Jean-Marie

    2014-05-15

    A new radioiodinated photoaffinity compound, [(125)I]YE(Bpa)WSLAAPQRFNH2, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (KD=0.24nM) with a single class of binding sites. It was also able to affinity label NPFF2 receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [(125)I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF2 receptor with apparent molecular weights of 140 and 95kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF2 receptor is observed. [(125)I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF2 receptors in vitro.

  7. High affinity germinal center B cells are actively selected into the plasma cell compartment.

    Science.gov (United States)

    Phan, Tri Giang; Paus, Didrik; Chan, Tyani D; Turner, Marian L; Nutt, Stephen L; Basten, Antony; Brink, Robert

    2006-10-30

    A hallmark of T cell-dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this "quality control" over plasma cell differentiation is likely critical for establishing effective humoral immunity.

  8. Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Christopher J Arnusch

    Full Text Available 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.

  9. Function and Regulation of the Plant COPT Family of High-Affinity Copper Transport Proteins

    Directory of Open Access Journals (Sweden)

    Sergi Puig

    2014-01-01

    Full Text Available Copper (Cu is an essential micronutrient for all eukaryotes because it participates as a redox active cofactor in multiple biological processes, including mitochondrial respiration, photosynthesis, oxidative stress protection, and iron (Fe transport. In eukaryotic cells, Cu transport toward the cytoplasm is mediated by the conserved CTR/COPT family of high-affinity Cu transport proteins. This outlook paper reviews the contribution of our research group to the characterization of the function played by the Arabidopsis thaliana COPT1–6 family of proteins in plant Cu homeostasis. Our studies indicate that the different tissue specificity, Cu-regulated expression, and subcellular localization dictate COPT-specialized contribution to plant Cu transport and distribution. By characterizing lack-of-function Arabidopsis mutant lines, we conclude that COPT1 mediates root Cu acquisition, COPT6 facilitates shoot Cu distribution, and COPT5 mobilizes Cu from storage organelles. Furthermore, our work with copt2 mutant and COPT-overexpressing plants has also uncovered Cu connections with Fe homeostasis and the circadian clock, respectively. Future studies on the interaction between COPT transporters and other components of the Cu homeostasis network will improve our knowledge of plant Cu acquisition, distribution, regulation, and utilization by Cu-proteins.

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

    Science.gov (United States)

    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.

  11. Lead, calcium uptake, and related genetic variants in association with renal cell carcinoma risk in a cohort of male Finnish smokers.

    Science.gov (United States)

    Southard, Emily B; Roff, Alanna; Fortugno, Tracey; Richie, John P; Kaag, Matthew; Chinchilli, Vernon M; Virtamo, Jarmo; Albanes, Demetrius; Weinstein, Stephanie; Wilson, Robin Taylor

    2012-01-01

    Lead is classified as a probable human carcinogen. However, its role in renal cell cancer (RCC) has not been established. Calcium and vitamin D may off-set toxicity in vivo. In this nested case-control study, whole blood lead, total serum calcium, and serum 25-hydroxyvitamin D levels were measured in blood drawn prior to diagnosis among male smokers participating in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Single-nucleotide polymorphisms (SNP) in five genes (CALB1, TRPV5, TRPV6, VDR, and ALAD) related to lead toxicity or calcium transport were genotyped. Logistic and linear regressions were used to determine RCC risk and time to diagnosis (respectively), adjusting for other risk factors. Among 154 newly diagnosed cases and 308 matched controls, RCC was associated with higher whole blood lead [OR = 2.0; 95% confidence interval (CI), 1.0-3.9; quartile 4 (Q4) vs. Q1, P(trend) = 0.022] and CALB1 rs1800645 (P(trend) = 0.025, minor 'T' allele frequency = 0.34). Higher total serum calcium (P(trend) ≤ 0.001) was associated with reduced RCC risk. Total serum calcium and 25-hydroxyvitamin D levels did not alter the association observed with lead. Time from enrollment to RCC diagnosis was positively associated with serum calcium (P(trend) = 0.002) and 25-hydroxyvitamin D (P(trend) = 0.054) among cases. Higher blood lead concentrations, below the 10 μg/dL level of concern, were associated with RCC, independent from serum calcium and CALB1 promoter polymorphism. Increased risk of RCC is associated with lower serum calcium and higher whole blood lead in smokers. The clinical prognostic value of serum calcium and vitamin D in RCC should be further investigated.

  12. Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj;

    2006-01-01

    Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells...

  13. Novel high-affinity and selective biaromatic 4-substituted ¿-hydroxybutyric acid (GHB) analogues as GHB ligands

    DEFF Research Database (Denmark)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte;

    2008-01-01

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites...

  14. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)prop...

  15. High-affinity accumulation of a maytansinoid in cells via weak tubulin interaction.

    Science.gov (United States)

    Goldmacher, Victor S; Audette, Charlene A; Guan, Yinghua; Sidhom, Eriene-Heidi; Shah, Jagesh V; Whiteman, Kathleen R; Kovtun, Yelena V

    2015-01-01

    The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.

  16. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors.

    Science.gov (United States)

    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.

  17. Devices and approaches for generating specific high-affinity nucleic acid aptamers

    Science.gov (United States)

    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.

  18. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    Energy Technology Data Exchange (ETDEWEB)

    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 (NIH); (Kansas); (HWMRI)

    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.

  19. A pharmacological profile of the high-affinity GluK5 kainate receptor.

    Science.gov (United States)

    Møllerud, Stine; Kastrup, Jette Sandholm; Pickering, Darryl S

    2016-10-05

    Mouse GluK5 was expressed in Sf9 insect cells and radiolabelled with [(3)H]-kainate in receptor binding assays (Kd=6.9nM). Western immunoblotting indicated an Sf9 GluK5 band doublet corresponding to the glycosylated (128kDa) and deglycosylated (111kDa) protein, which was identical to the band pattern of native rat brain GluK5. A pharmacological profile of the high-affinity kainate receptor GluK5 is described which is distinct from the profiles of other kainate receptors (GluK1-3). The 27 tested ligands generally show a preferential affinity to GluK1 over GluK5, the exceptions being: dihydrokainate, (S)-5-fluorowillardiine, (S)-glutamate and quisqualate, where the affinity is similar at GluK1 and GluK5. In contrast, quisqualate shows 40-fold higher affinity at GluK5 over GluK3 whereas (S)-1-(2'-amino-2'-caboxyethyl)thienol[3,4-d]pyrimidin-2,4-dione (NF1231), (RS)-2-amino-3-(5-tert-butyl-3-hydroxyisoxazol-4-yl)propionate (ATPA), dihydrokainate and (2S,4R)-4-methyl-glutamate (SYM2081) have higher affinity at GluK3 compared to GluK5. Since some studies have indicated that GluK5 is associated with various diseases in the central nervous system (e.g. schizophrenia, temporal lobe epilepsy, bipolar disorder), selective GluK5 ligands could have therapeutic potential. The distinct pharmacological profile of GluK5 suggests that it would be possible to design ligands with selectivity towards GluK5.

  20. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    Directory of Open Access Journals (Sweden)

    Klaus B Nissen

    Full Text Available 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.

  1. In vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers.

    Science.gov (United States)

    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.

  2. The C2 domains of granuphilin are high-affinity sensors for plasma membrane lipids.

    Science.gov (United States)

    Lyakhova, Tatyana A; Knight, Jefferson D

    2014-09-01

    Membrane-targeting proteins are crucial components of many cell signaling pathways, including the secretion of insulin. Granuphilin, also known as synaptotagmin-like protein 4, functions in tethering secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin docks to insulin secretory vesicles through interaction of its N-terminal domain with vesicular Rab proteins; however, the mechanisms of granuphilin plasma membrane targeting and release are less clear. Granuphilin contains two C2 domains, C2A and C2B, that interact with the plasma membrane lipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The goal of this study was to determine membrane-binding mechanisms, affinities, and kinetics of both granuphilin C2 domains using fluorescence spectroscopic techniques. Results indicate that both C2A and C2B bind anionic lipids in a Ca(2+)-independent manner. The C2A domain binds liposomes containing a physiological mixture of lipids including 2% PI(4,5)P2 or PI(3,4,5)P3 with high affinity (apparent K(d, PIPx) of 2-5 nM), and binds nonspecifically with moderate affinity to anionic liposomes lacking phosphatidylinositol phosphate (PIPx) lipids. The C2B domain binds with sub-micromolar affinity to liposomes containing PI(4,5)P2 but does not have a measurable affinity for background anionic lipids. Both domains can be competed away from their target lipids by the soluble PIPx analog inositol-(1,2,3,4,5,6)-hexakisphosphate (IP6), which is a positive regulator of insulin secretion. Potential roles of these interactions in the docking and release of granuphilin from the plasma membrane are discussed.

  3. Integrin alphaVbeta6 is a high-affinity receptor for coxsackievirus A9.

    Science.gov (United States)

    Heikkilä, Outi; Susi, Petri; Stanway, Glyn; Hyypiä, Timo

    2009-01-01

    Coxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins alphaVbeta3 and alphaVbeta6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins alphaVbeta3 and alphaVbeta6, whereas alphaVbeta6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized alphaVbeta3 and alphaVbeta6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against alphaV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble alphaVbeta6, but not alphaVbeta3, bound to immobilized CAV9. Similarly, only soluble alphaVbeta6 blocked virus infectivity. These data suggest that CAV9 binding to alphaVbeta6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined.

  4. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    Science.gov (United States)

    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

    2010-01-01

    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 Å 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. PMID:20194791

  5. In silico analysis of high affinity potassium transporter (HKT) isoforms in different plants.

    Science.gov (United States)

    Zamani Babgohari, Mahbobeh; Ebrahimie, Esmaeil; Niazi, Ali

    2014-01-01

    High affinity potassium transporters (HKTs) are located in the plasma membrane of the vessels and have significant influence on salt tolerance in some plants. They exclude Na(+) from the parenchyma cells to reduce Na(+) concentration. Despite many studies, the underlying regulatory mechanisms and the exact functions of HKTs within different genomic backgrounds are relatively unknown. In this study, various bioinformatics techniques, including promoter analysis, identification of HKT-surrounding genes, and construction of gene networks, were applied to investigate the HKT regulatory mechanism. Promoter analysis showed that rice HKTs carry ABA response elements. Additionally, jasmonic acid response elements were detected on promoter region of TmHKT1;5. In silico synteny highlighted several unknown and new loci near rice, Arabidopsis thaliana and Physcomitrella patent HKTs, which may play a significant role in salt stress tolerance in concert with HKTs. Gene network prediction unravelled that crosstalk between jasmonate and ethylene reduces AtHKT1;1 expression. Furthermore, antiporter and transferase proteins were found in AtHKT1;1 gene network. Interestingly, regulatory elements on the promoter region of HKT in wild genotype (TmHKT1;5) were more frequent and variable than the ones in cultivated wheat (TaHKT1;5) which provides the possibility of rapid response and better understanding of environmental conditions for wild genotype. Detecting ABA and jasmonic acid response elements on promoter regions of HKTs provide valuable clues on underlying regulatory mechanisms of HKTs. In silico synteny and pathway discovery indicated several candidates which act in concert with HKTs in stress condition. We highlighted different arrangement of regulatory elements on promoter region of wild wheat (TmHKT1;5) compared to bread wheat (TaHKT1;5) in this study.

  6. Novel and high affinity fluorescent ligands for the serotonin transporter based on (s)-citalopram

    DEFF Research Database (Denmark)

    Kumar, Vivek; Rahbek-Clemmensen, Troels; Billesbølle, Christian B

    2014-01-01

    Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently transfected COS7 cells. Compound 14 demons...

  7. The diamidine diminazene aceturate is a substrate for the high-affinity pentamidine transporter: implications for the development of high resistance levels in trypanosomes.

    Science.gov (United States)

    Teka, Ibrahim A; Kazibwe, Anne J N; El-Sabbagh, Nasser; Al-Salabi, Mohammed I; Ward, Christopher P; Eze, Anthonius A; Munday, Jane C; Mäser, Pascal; Matovu, Enock; Barrett, Michael P; de Koning, Harry P

    2011-07-01

    African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(-/-), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The K(m) for diminazene transport in bloodstream tbat1(-/-) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(-/-), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes.

  8. Taking Advantage: High Affinity B cells in the Germinal Center Have Lower Death Rates, But Similar Rates of Division Compared to Low Affinity Cells1

    OpenAIRE

    2009-01-01

    B lymphocytes producing high affinity antibodies (Abs) are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high affinity B cells are selected during the immune response has never been elucidated. Though it has been shown that high affinity cells directly outcompete low affinity cells in the germinal center (GC)2, whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity c...

  9. Structures of the ultra-high-affinity protein–protein complexes of pyocins S2 and AP41 and their cognate immunity proteins from pseudomonas aeruginosa

    OpenAIRE

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna; Lowe, Edward; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander; Cogdell, Richard; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despit...

  10. Adjusting ammonium uptake via phosphorylation.

    Science.gov (United States)

    Lanquar, Viviane; Frommer, Wolf B

    2010-06-01

    In plants, AMT/MEP/Rh superfamily mediates high affinity ammonium uptake. AMT/MEP transporters form a trimeric complex, which requires a productive interaction between subunits in order to be functional. The AMT/MEP C-terminal domain is highly conserved in more than 700 AMT homologs from cyanobacteria to higher plants with no cases found to be lacking this domain. AMT1;1 exists in active and inactive states, probably controlled by the spatial positioning of the C-terminus. Ammonium triggers the phosphorylation of a conserved threonine residue (T460) in the C-terminus of AMT1;1 in a time- and concentration-dependent manner. The T460 phosphorylation level correlates with a decrease of root ammonium uptake. We propose that ammonium-induced phosphorylation modulates ammonium uptake as a general mechanism to protect against ammonium toxicity.

  11. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Identification of a high-affinity ligand that exhibits complete aryl hydrocarbon receptor antagonism.

    Science.gov (United States)

    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

  13. Structural characterization of a high affinity mononuclear site in the copper(II)-α-synuclein complex.

    Science.gov (United States)

    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.

  14. Inhibition of the high affinity choline transporter enhances hyperalgesia in a rat model of chronic pancreatitis.

    Science.gov (United States)

    Luo, Dan; Chen, Lei; Yu, Baoping

    2017-06-17

    The mechanisms underlying chronic and persistent pain associated with chronic pancreatitis (CP) are not completely understood. The cholinergic system is one of the major neural pathways of the pancreas. Meanwhile, this system plays an important role in chronic pain. We hypothesized that the high affinity choline transporter CHT1, which is a main determinant of cholinergic signaling capacity, is involved in regulating pain associated with CP. CP was induced by intraductal injection of 2% trinitrobenzene sulfonic acid (TNBS) in Sprague-Dawley rats. Pathological examination was used to evaluate the inflammation of pancreas and hyperalgesia was assessed by measuring the number of withdrawal events evoked by application of the von Frey filaments. CHT1 expression in pancreas-specific dorsal root ganglia (DRGs) was assessed through immunohistochemistry and western blotting. We also intraperitoneally injected the rats with hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Then we observed its effects on the visceral hyperalgesia induced by CP, and on the acetylcholine (ACh) levels in the DRGs through using an acetylcholine/acetylcholinesterase assay kit. Signs of CP were observed 21 days after TNBS injection. Rats subjected to TNBS infusions had increased sensitivity to mechanical stimulation of the abdomen. CHT1-immunoreactive cells were increased in the DRGs from rats with CP compared to naive or sham rats. Western blots indicated that CHT1 expression was significantly up-regulated in TNBS-treated rats when compared to naive or sham-operated rats at all time points following surgery. In the TNBS group, CHT1 expression was higher on day 28 than on day 7 or day 14, but there was no statistical difference in CHT1 expression on day 28 vs. day 21. Treatment with HC-3 (60 μg/kg, 80 μg/kg, or 100 μg/kg) markedly enhanced the mechanical hyperalgesia and reduced ACh levels in a dose-dependent manner in rats with CP. We report for the first time that CHT1 may be involved

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

    Science.gov (United States)

    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.

  16. In vitro characterization of cadmium and zinc uptake via the gastro-intestinal tract of the rainbow trout (Oncorhynchus mykiss): Interactive effects and the influence of calcium

    Energy Technology Data Exchange (ETDEWEB)

    Ojo, Adeola A. [Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1 (Canada)], E-mail: adeolaojo25@yahoo.com; Wood, Chris M. [Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1 (Canada)], E-mail: woodcm@mcmaster.ca

    2008-08-11

    An in vitro gut sac technique was employed to study whether Cd and Zn uptake mechanisms in the gastro-intestinal tract of the rainbow trout are similar to those at the gills, where both metals are taken up via the Ca transport pathway. Metal accumulation in surface mucus, in the mucosal epithelium, and transport into the blood space were assayed using radiolabelled Cd or Zn concentrations of 50 {mu}mol L{sup -1} in the luminal (internal) saline. Elevated luminal Ca (10 or 100 mmol L{sup -1}versus 1 mmol L{sup -1}) reduced Cd uptake into all three phases by approximately 60% in the stomach, but had no effect in the anterior, mid, or posterior intestine. This finding is in accordance with recent in vivo evidence that Ca is taken up mainly via the stomach, and that high [Ca] diets inhibit Cd accumulation from the food specifically in this section of the tract. In contrast, 10 mmol L{sup -1} luminal Ca had no effect on Zn transport in any section, whereas 100 mmol L{sup -1} Ca stimulated Zn uptake, by approximately threefold, into all three phases in the stomach only. There was no influence of elevated luminal Zn (10 mmol L{sup -1}) on Cd uptake in the stomach or anterior intestine, or of high Cd (10 mmol L{sup -1}) on Zn uptake in these sections. However, high [Zn] stimulated Cd transport into the blood space but inhibited accumulation in the mucosal epithelium and/or mucus-binding in the mid and posterior intestine, whereas high [Cd] exerted a reciprocal effect in the mid-intestine only. We conclude that Cd uptake occurs via an important Ca-sensitive mechanism in the stomach which is different from that at the gills, while Cd transport mechanisms in the intestine are not directly Ca-sensitive. Zn uptake does not appear to involve Ca uptake pathways, in contrast to the gills. These results are discussed in the context of other possible Cd and Zn transport pathways, and the emerging role of the stomach as an organ of divalent metal uptake.

  17. PREPARATION OF IMMUNOGEN AND PURIFICA¬TION OF HIGH AFFINITY AND SPECIFICITY FAB FRAGMENT OF ANTI-DIGOXIN POLYCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    M. Pour-Amir

    2000-01-01

    Full Text Available In this study we produced and purified a high titer of specific and high affin¬ity Fab fragments of anti-digoxin antibody. Immunization of rabbits with a conju¬gate of the cardiac glycoside digoxin, coupled by a periodate oxidation method to the amino group of lysine in bovine serum albumin resulted in the production of this type of high titer digoxin-specific antibodies with exceptionally high affinity (109 L/mol and specificity in immune response. Increase in titer was found in steps of purification ending up with the highest titer for Fab fragment to be at 1.75 ug of purified Fab (for 50% binding of I25I-digoxin. High specificity for antigenic determinants of the steroid nucleus of digoxin was observed such that much less cross-reaction with digoxin (2.3% and no cross-reaction with ouabaine, estradiol, Cortisol, progesterone and testosterone were detected.

  18. Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

    Science.gov (United States)

    Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

    2015-09-01

    Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

  19. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA

    DEFF Research Database (Denmark)

    Klein, A B; Bay, T; Villumsen, I S

    2016-01-01

    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...

  20. Breathing air to save energy--new insights into the ecophysiological role of high-affinity [NiFe]-hydrogenase in Streptomyces avermitilis.

    Science.gov (United States)

    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.

  1. Inhibition of human high-affinity copper importer Ctr1 orthologous in the nervous system of Drosophila ameliorates Aβ42-induced Alzheimer's disease-like symptoms.

    Science.gov (United States)

    Lang, Minglin; Fan, Qiangwang; Wang, Lei; Zheng, Yajun; Xiao, Guiran; Wang, Xiaoxi; Wang, Wei; Zhong, Yi; Zhou, Bing

    2013-11-01

    Disruption of copper homeostasis has been implicated in Alzheimer's disease (AD) during the last 2 decades; however, whether copper is a friend or a foe is controversial. Within a genetically tractable Drosophila AD model, we manipulated the expression of human high-affinity copper importer orthologous in Drosophila to explore the in vivo roles of copper ions in the development of AD. We found that inhibition of Ctr1C expression by RNAi in Aβ-expressing flies significantly reduced copper accumulation in the brains of the flies as well as ameliorating neurodegeneration, enhancing climbing ability, and prolonging lifespan. Interestingly, Ctr1C inhibition led to a significant increase in higher-molecular-weight Aβ42 forms in brain lysates, whereas it was accompanied by a trend of decreased expression of amyloid-β degradation proteases (including NEP1-3 and IDE) with age and reduced Cu-Aβ interaction-induced oxidative stress in Ctr1C RNAi flies. Similar results were obtained from inhibiting another copper importer Ctr1B and overexpressing a copper exporter DmATP7 in the nervous system of AD flies. These results imply that copper may play a causative role in developing AD, as either Aβ oligomers or aggregates were less toxic in a reduced copper environment or one with less copper binding. Early manipulation of brain copper uptake can have a great effect on Aβ pathology.

  2. Soluble T cell receptor Vβ domains engineered for high-affinity binding to staphylococcal or streptococcal superantigens.

    Science.gov (United States)

    Sharma, Preeti; Wang, Ningyan; Kranz, David M

    2014-01-28

    Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs), so-called because they stimulate a large fraction of an individual's T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR) on a T cell and a class II product of the major histocompatibility complex (MHC) on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V) of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1) or S. pyogenes (SpeA and SpeC) have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands.

  3. Immunotherapy Expands and Maintains the Function of High-Affinity Tumor-Infiltrating CD8 T Cells In Situ.

    Science.gov (United States)

    Moran, Amy E; Polesso, Fanny; Weinberg, Andrew D

    2016-09-15

    Cancer cells harbor high-affinity tumor-associated Ags capable of eliciting potent antitumor T cell responses, yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and programmed death-1 (PD-1) have been used. We report in this study that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor Ag-specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Coexpression of Nur77GFP and OX40 identifies a polyclonal population of high-affinity tumor-associated Ag-specific CD8(+) T cells, which produce more IFN-γ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or programmed death ligand-1. Moreover, expansion of these high-affinity CD8 T cells prolongs survival of tumor-bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired, and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor-resident CD8 T cells, thereby increasing the frequency of cytotoxic, high-affinity, tumor-associated Ag-specific cells.

  4. α4βδ GABA receptors are high-affinity targets for γ-hydroxybutyric acid (GHB)

    DEFF Research Database (Denmark)

    Absalom, N.; Karim, N.; Eghorn, L.F.;

    2012-01-01

    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-affinit...... 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....

  5. Soluble T Cell Receptor Vβ Domains Engineered for High-Affinity Binding to Staphylococcal or Streptococcal Superantigens

    Directory of Open Access Journals (Sweden)

    Preeti Sharma

    2014-01-01

    Full Text Available Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs, so-called because they stimulate a large fraction of an individual’s T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR on a T cell and a class II product of the major histocompatibility complex (MHC on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1 or S. pyogenes (SpeA and SpeC have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands.

  6. Effect of broiler litter ash and flue gas desulfurization gypsum on yield, calcium, phosphorus, copper, iron, manganese and zinc uptake by peanut

    Science.gov (United States)

    Peanut (Arachis hyogaea) is an important oil seed crop that is grown as a principle source of edible oil and vegetable protein. Over 1.6 million acres of peanuts were planted in the United States during 2012. Peanuts require large amounts of calcium (Ca) and phosphorus (P). In 2010, over 10 milli...

  7. Analysis of high affinity self-association by fluorescence optical sedimentation velocity analytical ultracentrifugation of labeled proteins: opportunities and limitations.

    Directory of Open Access Journals (Sweden)

    Huaying Zhao

    Full Text Available Sedimentation velocity analytical ultracentrifugation (SV is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer species. Specifically, FAM (5,6-carboxyfluorescein was found to create different populations of artificially high-affinity and low-affinity dimers, as indicated by both FDS-SV and the kinetics of dimer dissociation studied using a bench-top fluorescence spectrometer and Förster Resonance Energy Transfer. By contrast, Dylight488 labeled GluA2, as well as GluA2 expressed as an EGFP fusion protein, yielded results consistent with estimates for unlabeled GluA2. Our study suggests considerations for the choice of labeling strategies, and highlights experimental designs that exploit specific opportunities of FDS-SV for improving the reliability of the binding isotherm analysis of interacting systems.

  8. High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

    Science.gov (United States)

    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.

  9. Expression of Brassica napus L. γ-Glutamylcysteine Synthetase and Low-and High-Affinity Sulfate Transporters in Response to Excess Cadmium

    Institute of Scientific and Technical Information of China (English)

    Xin SUN; Xue-Mei SUN; Zhi-Min YANG; Shao-Qiong LI; Jin WANG; Song-Hua WANG

    2005-01-01

    In both the roots and leaves ofBrassica napus L. cv. Youyan No. 8 under treatment with 30 μmol/L Cd, massive production of non-protein thiols (NPT; mainly containing glutathione (GSH) and phytochelatins (PCs)) was induced, together with an increase in γ-glutamylcysteine synthetase (γ-ECS)mRNA transcripts. Because γ-ECS is the key enzyme catalyzing the first step in GSH biosynthesis, which, in turn, is converted to PCs, the Cd-induced increase in γ-ECS expression may be responsible for the observed increase in the production of NPT. Using a quantitative reverse transcription polymerase chain reaction (RT-PCR) approach, the expression of genes encoding a putative low-affinity sulfate transporter (LAST) and a putative high-affinity sulfate transporter (HAST) was determined at the transcriptional level. The RT-PCR analysis of relative transcript amounts indicates that the LAST gene in B. napus leaves showed a constitutive expression, which was hardly affected by Cd treatment. However, treatment with 30 μmol/L Cd for 2 or 3 d induced a marked increase in the expression of LAST in roots. Transcriptional expression of the HAST gene occurred in roots, but not in leaves. The expression of HAST only in the roots suggests that it has a specific function in sulfate uptake from soil and that the putative LAST may be responsible for the transport of sulfate from the roots to the shoots, as well as for the uptake of sulfate from soil. These results indicate that changes in transcriptional expression for sulfate transporters were required for the increased demand for sulfate during Cd stress.

  10. Assembly, activation, and trafficking of the Fet3p.Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae.

    Science.gov (United States)

    Singh, Arvinder; Severance, Scott; Kaur, Navjot; Wiltsie, William; Kosman, Daniel J

    2006-05-12

    The high affinity iron uptake complex in the yeast plasma membrane (PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two-hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer (FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptake-competent complex. The cytoplasmic, carboxyl-terminal domain of each protein contains a four-residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two-hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the approximately 13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain (TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl-terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease (monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a (minimal) heterodimer model of the Fet3p.Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  13. Uptake and caffeine-induced release of calcium in fast muscle fibers of Xenopus laevis: effects of MgATP and P(i).

    Science.gov (United States)

    Stienen, G J; van Graas, I A; Elzinga, G

    1993-09-01

    To elucidate the origin of the reduction in force during prolonged muscle fatigue, the dependency of Ca2+ uptake and release on MgATP and P(i) concentration was studied in saponin-skinned fast skeletal muscle fibers of the iliofibularis muscle of Xenopus laevis at 3 degrees C. The sarcoplasmic reticulum was loaded with Ca2+ for 5 min at pCa 7.0. The amount of Ca2+ released was derived from the area of the caffeine-induced force response. Ca2+ uptake increased with the MgATP concentration present during loading. It was half maximal at 20 microM and saturated at higher concentrations. The kinetics of Ca2+ release were affected for MgATP concentrations between 0.1 and 0.5 mM or less, but the amount of Ca2+ released by caffeine in ATP-free solutions was substantial. Phosphate (15 mM) only slightly reduced Ca2+ uptake when the loading period was short (1 min). It is unlikely, therefore, that the reduction in MgATP concentration contributes to the depression of Ca2+ released from the sarcoplasmic reticulum during fatigue. The increase in P(i) concentration could play a small role by reducing Ca2+ uptake.

  14. Sodium-dependent phosphate uptake in the jejunum is post-transcriptionally regulated in pigs fed a low-phosphorus diet and is independent of dietary calcium concentration.

    Science.gov (United States)

    Saddoris, Kari L; Fleet, James C; Radcliffe, John S

    2010-04-01

    In rodents, severe dietary P restriction increases active phosphate absorption by the intestine. However, it remains unknown if moderate dietary P restriction has a similar effect. Weanling pigs (n = 32; body weight 7.4 +/- 0.55 kg) were used in a 2 x 2 factorial design and fed dietary available P (aP) concentrations of 0.23 or 0.40% and Ca concentrations of 0.58 or 1.00% for 14 d. Diets were formulated on an aP basis instead of a total P basis, because pigs are unable to absorb phytate-P present in corn and soybean meal. Jejunal segments were mounted in modified Ussing chambers for determination of Na(+)-dependent nutrient transport. Intestinal mucosal scrapings were taken for RNA isolation and brush border membrane (BBM) vesicle isolation. Na(+)-dependent phosphate uptake and gene expression of Na-phosphate cotransporter IIb (NaPi-IIb), SGLT-1 (sodium/glucose cotransporter-1), and calbindin D(9k) and protein expression of NaPi-IIb were evaluated. Na(+)-dependent phosphate transport increased (P dietary aP concentration was decreased. However, increased Na(+)-dependent phosphate uptake was not accompanied by increased NaPi-IIb mRNA expression. Expression of NaPi-IIb protein in the BBM increased (P pigs fed low-P diets compared with pigs fed adequate-P diets. No dietary Ca effects or aP x Ca interactions were detected for Na-dependent P uptake, mRNA or protein expression of NaPi-IIb, or mRNA expression of calbindin D(9k). These data suggest that restricting dietary aP concentration by only 43% stimulates Na(+)-dependent phosphate uptake and expression of the NaPi-IIb protein in the BBM of the small intestine and through a post-transcriptional mechanism.

  15. Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes

    Directory of Open Access Journals (Sweden)

    Dmitry eSamigullin

    2015-01-01

    Full Text Available At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 рА and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 µM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

  16. α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

    Science.gov (United States)

    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.

  17. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA.

    Science.gov (United States)

    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.

  18. Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

    Science.gov (United States)

    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 Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

  19. (/sup 3/H)pirenzepine selectively identifies a high affinity population of muscarinic cholinergic receptors in the rat cerebral cortex

    Energy Technology Data Exchange (ETDEWEB)

    Watson, M.; Roeske, W.R.; Yamamura, H.I.

    1982-11-01

    The specific binding of (/sup 3/H)pirenzepine was investigated in homogenates of rat cerebral cortex, cerebellar cortex, and heart. Specific binding of (/sup 3/H)pirenzepine in the cerebral cortex as defined by displacement with atropine sulfate (1..mu..M) was of high affinity (K/sub d/ = 4-10 nM, receptor density = 1.06 pmoles/mg protein), stereoselective, and competitive with drugs specific for the muscarinic receptor. In contrast, few (/sup 3/H)pirenzepine binding sites were demonstrated in cerebellar and heart homogenates.

  20. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

    OpenAIRE

    Liu, F T; Albrandt, K; Robertson, M W

    1988-01-01

    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, w...

  1. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin.

    Science.gov (United States)

    Ryan, Ali J; Chung, Chun-Wa; Curry, Stephen

    2011-04-18

    Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA). It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which--in drugs sites 1 and 2 on the protein--are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.

  2. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin

    Directory of Open Access Journals (Sweden)

    Chung Chun-wa

    2011-04-01

    Full Text Available Abstract Background Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA. It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. Results We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which - in drugs sites 1 and 2 on the protein - are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. Conclusions The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.

  3. (TH)205-501, a non-catechol dopaminergic agonist, labels selectively and with high affinity dopamine D2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Closse, A.; Frick, W.; Markstein, R.; Maurer, R.; Nordmann, R.

    1985-01-01

    (TH)205-501, a non dopaminergic agonist, is presented as a ligand with high affinity (Ksub(D) approx= 1 nM) and high selectivity for dopamine receptors. pKsubi values of dopaminergic agonists derived from competition isotherms in the (TH)205-501 binding assay correlate very well with their potency in the acetylcholine release assay, which is controlled by dopamine D2 receptors. There is however no correlation with their potency stimulating aldenylate cyclase, a process controlled by dopamine D1 receptors. Thus (TH)205-501 is the first agonist ligand selective for dopamine D2 receptors. (Author).

  4. Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

    Energy Technology Data Exchange (ETDEWEB)

    Steingroewer, Juliane [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany)]. E-mail: juliane.steingroewer@tu-dresden.de; Bley, Thomas [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany); Bergemann, Christian [Chemicell GmbH, D-10823, Berlin (Germany); Boschke, Elke [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany)

    2007-04-15

    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.

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

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida

    2015-01-01

    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...... 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...

  6. Activation-induced spatiotemporal cerebral blood flow changes and behavioral deficit after developmental mTBI in rats can be favorably altered by facilitating mitochondrial calcium uptake

    Directory of Open Access Journals (Sweden)

    Madhuvika eMurugan

    2016-03-01

    Full Text Available Mild to moderate traumatic brain injury (mTBI leads to secondary neuronal loss via excitotoxic mechanisms, including mitochondrial Ca2+ overload. However in the surviving cellular population, mitochondrial Ca2+ influx and oxidative metabolism are diminished leading to suboptimal neuronal circuit activity and poor prognosis. Hence we tested the impact of boosting neuronal electrical activity and oxidative metabolism by facilitating mitochondrial Ca2+ uptake in a rat model of mTBI. In developing rats (P25-P26 sustaining an mTBI, we demonstrate post-traumatic changes in cerebral blood flow (CBF in the sensorimotor cortex in response to whisker stimulation compared to sham using functional Laser Doppler Imaging (fLDI at adulthood (P67-P73. Compared to sham, whisker stimulation-evoked positive CBF responses decreased while negative CBF responses increased in the mTBI animals. The spatiotemporal CBF changes representing underlying neuronal activity suggested profound changes to neurovascular activity after mTBI. Behavioral assessment of the same cohort of animals prior to fLDI showed that mTBI resulted in persistent contralateral sensorimotor behavioral deficit along with ipsilateral neuronal loss compared to sham. Treating mTBI rats with Kaempferol, a dietary flavonol compound that enhanced mitochondrial Ca2+ uptake, eliminated the inter-hemispheric asymmetry in the whisker stimulation-induced positive CBF responses and the ipsilateral negative CBF responses otherwise observed in the untreated and vehicle-treated mTBI animals in adulthood. Kaempferol also improved somatosensory behavioral measures compared to untreated and vehicle treated mTBI animals without augmenting post-injury neuronal loss. The results indicate that reduced mitochondrial Ca2+ uptake in the surviving populations affect post-traumatic neural activation leading to persistent behavioral deficits. Improvement in sensorimotor behavior and spatiotemporal neurovascular activity

  7. High-affinity hemoglobin and blood oxygen saturation in diving emperor penguins.

    Science.gov (United States)

    Meir, Jessica U; Ponganis, Paul J

    2009-10-01

    The emperor penguin (Aptenodytes forsteri) thrives in the Antarctic underwater environment, diving to depths greater than 500 m and for durations longer than 23 min. To examine mechanisms underlying the exceptional diving ability of this species and further describe blood oxygen (O2) transport and depletion while diving, we characterized the O2-hemoglobin (Hb) dissociation curve of the emperor penguin in whole blood. This allowed us to (1) investigate the biochemical adaptation of Hb in this species, and (2) address blood O2 depletion during diving, by applying the dissociation curve to previously collected partial pressure of O2 (PO2) profiles to estimate in vivo Hb saturation (SO2) changes during dives. This investigation revealed enhanced Hb-O2 affinity (P50=28 mmHg, pH 7.5) in the emperor penguin, similar to high-altitude birds and other penguin species. This allows for increased O2 at low blood PO2 levels during diving and more complete depletion of the respiratory O2 store. SO2 profiles during diving demonstrated that arterial SO2 levels are maintained near 100% throughout much of the dive, not decreasing significantly until the final ascent phase. End-of-dive venous SO2 values were widely distributed and optimization of the venous blood O2 store resulted from arterialization and near complete depletion of venous blood O2 during longer dives. The estimated contribution of the blood O2 store to diving metabolic rate was low and highly variable. This pattern is due, in part, to the influx of O2 from the lungs into the blood during diving, and variable rates of tissue O2 uptake.

  8. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Sherman, S.J.; Catterall, W.A.

    1982-11-01

    Specific binding of /sup 3/H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors.

  9. Identification of a high-affinity Ca sup 2+ pump associated with endocytotic vesicles in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Milne, J.L.; Coukell, M.B. (York Univ., North York, Ontario (Canada))

    1989-11-01

    In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca{sup 2+} are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca{sup 2+} levels in this organism, the authors previously isolated and characterized an ATP/Mg{sup 2+}-dependent, high-affinity Ca{sup 2+} pump which appeared to be a component of inside-out plasma membrane vesicles. In this report, they demonstrate that a high-affinity Ca{sup 2+} pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca{sup 2+}-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca{sup 2+} transporter is associated with a high-capacity non-IP{sub 3}-releasable Ca{sup 2+} store which is generated by endocytosis. A possible role for this store in maintaining Ca{sup 2+} homeostasis in Dictyostelium is discussed.

  10. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

    Science.gov (United States)

    Liu, F T; Albrandt, K; Robertson, M W

    1988-08-01

    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.

  11. Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay.

    Science.gov (United States)

    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.

  12. The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate.

    Science.gov (United States)

    Kristensen, Ole; Kristensen, Lise Baadsgaard; Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm

    2016-09-01

    Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

  13. Modulation of neuronal serotonin uptake by a putative endogenous ligand of imipramine recognition sites.

    OpenAIRE

    Barbaccia, M. L.; Gandolfi, O; Chuang, D M; Costa, E

    1983-01-01

    Imipramine inhibits the serotonin uptake by binding with high affinity to regulatory sites of this uptake located on axons that release serotonin. The number of imipramine recognition sites located on crude synaptic membrane preparations is reduced by two daily injections of imipramine or desmethylimipramine for 3 weeks. When the binding sites for [3H]imipramine are down-regulated the Vmax of the neuronal uptake of serotonin is increased. Moreover, in minces prepared from the brain hippocampu...

  14. Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo

    National Research Council Canada - National Science Library

    Yu, Channing; Cantor, Alan B; Yang, Haidi; Browne, Carol; Wells, Richard A; Fujiwara, Yuko; Orkin, Stuart H

    2002-01-01

    .... Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss...

  15. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

    DEFF Research Database (Denmark)

    Eghorn, Laura Friis; Høstgaard-Jensen, Kirsten; Kongstad, Kenneth Thermann

    2014-01-01

    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......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...

  16. Salicylic acid alleviates aluminum toxicity in rice seedlings better than magnesium and calcium by reducing aluminum uptake, suppressing oxidative damage and increasing antioxidative defense.

    Science.gov (United States)

    Pandey, Poonam; Srivastava, Rajneesh Kumar; Dubey, R S

    2013-05-01

    Aluminum toxicity is a major constraint to crop production in acid soils. The present study was undertaken to examine the comparative ameliorating effects of salicylic acid, Ca and Mg on Al toxicity in rice (Oryza sativa L.) seedlings grown in hydroponics. Al treatment (0.5 mM AlCl3) caused decrease in plant vigour, loss of root plasma membrane integrity, increased contents of O 2 (∙-) , H2O2, lipid peroxidation, protein carbonyls and decline in the level of protein thiol. Al treatment caused significant changes in activity of antioxidative enzymes in rice seedlings. Exogenously added salicylic acid (60 μM), Ca (1 mM) and Mg (0.25 mM) significantly alleviated Al toxicity effects in the seedlings marked by restoration of growth, suppression of Al uptake, restoration of root plasma membrane integrity and decline in O 2 (∙-) , H2O2, lipid peroxidation and protein carbonyl contents. Salicylic acid, Ca and Mg suppressed Al-induced increase in SOD, GPX and APX activities while it elevated Al-induced decline in CAT activity. By histochemical staining of O 2 (∙-) using NBT and H2O2 using DAB, it was further confirmed that added salicylic acid, Ca or Mg decreased Al-induced accumulation of O 2 (∙-) and H2O2 in the leaf tissues. Results indicate that exogenously added salicylic acid, Ca or Mg alleviates Al toxicity in rice seedlings by suppressing Al uptake, restoring root membrane integrity, reducing ROS level and ROS induced oxidative damage and regulating the level of antioxidative enzyme activities. Further salicylic appears to be superior to Mg and Ca in alleviating Al toxicity effects in rice plants.

  17. 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.

    Science.gov (United States)

    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 ( F). For longissimus dorsi, EAAC1 ( 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 ( adipose, respectively, and not differing ( > 0.45) from omental or mesenchymal adipose. These data demonstrate (1) longissimus dorsi and adipose tissues of steers developing through typical

  18. Transport Properties of the Tomato Fruit Tonoplast : III. Temperature Dependence of Calcium Transport.

    Science.gov (United States)

    Joyce, D C; Cramer, G R; Reid, M S; Bennett, A B

    1988-12-01

    Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca(2+) transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca(2+). A low affinity Ca(2+) uptake system (K(m) > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H(+)/Ca(2+) antiport. A high affinity Ca(2+) uptake system (K(m) = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca(2+) transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12 degrees C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca(2+)/H(+) antiport activity could only by partially ascribed to an effect of low temperature on H(+)-ATPase activity, ATP-dependent H(+) transport, passive H(+) fluxes, or passive Ca(2+) fluxes. These results suggest that low temperature directly affects Ca(2+)/H(+) exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca(2+)/H(+) exchange protein or by an indirect effect of temperature on lipid interactions with the Ca(2+)/H(+) exchange protein.

  19. A ferroxidation/permeation iron uptake system is required for virulence in Ustilago maydis.

    Science.gov (United States)

    Eichhorn, Heiko; Lessing, Franziska; Winterberg, Britta; Schirawski, Jan; Kämper, Jörg; Müller, Philip; Kahmann, Regine

    2006-11-01

    In the smut fungus Ustilago maydis, a tightly regulated cAMP signaling cascade is necessary for pathogenic development. Transcriptome analysis using whole genome microarrays set up to identify putative target genes of the protein kinase A catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity iron uptake systems. These genes locate to three gene clusters on different chromosomes and include the previously identified complementing siderophore auxotroph genes sid1 and sid2 involved in siderophore biosynthesis. Transcription of all nine genes plus three additional genes associated with the gene clusters was also coregulated by iron through the Urbs1 transcription factor. Two components of a high-affinity iron uptake system were characterized in more detail: fer2, encoding a high-affinity iron permease; and fer1, encoding an iron multicopper oxidase. Fer2 localized to the plasma membrane and complemented an ftr1 mutant of Saccharomyces cerevisiae lacking a high-affinity iron permease. During pathogenic development, fer2 expression was confined to the phase of hyphal proliferation inside the plant. fer2 as well as fer1 deletion mutants were strongly affected in virulence. These data highlight the importance of the high-affinity iron uptake system via an iron permease and a multicopper oxidase for biotrophic development in the U. maydis/maize (Zea mays) pathosystem.

  20. A Ferroxidation/Permeation Iron Uptake System Is Required for Virulence in Ustilago maydis[W

    Science.gov (United States)

    Eichhorn, Heiko; Lessing, Franziska; Winterberg, Britta; Schirawski, Jan; Kämper, Jörg; Müller, Philip; Kahmann, Regine

    2006-01-01

    In the smut fungus Ustilago maydis, a tightly regulated cAMP signaling cascade is necessary for pathogenic development. Transcriptome analysis using whole genome microarrays set up to identify putative target genes of the protein kinase A catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity iron uptake systems. These genes locate to three gene clusters on different chromosomes and include the previously identified complementing siderophore auxotroph genes sid1 and sid2 involved in siderophore biosynthesis. Transcription of all nine genes plus three additional genes associated with the gene clusters was also coregulated by iron through the Urbs1 transcription factor. Two components of a high-affinity iron uptake system were characterized in more detail: fer2, encoding a high-affinity iron permease; and fer1, encoding an iron multicopper oxidase. Fer2 localized to the plasma membrane and complemented an ftr1 mutant of Saccharomyces cerevisiae lacking a high-affinity iron permease. During pathogenic development, fer2 expression was confined to the phase of hyphal proliferation inside the plant. fer2 as well as fer1 deletion mutants were strongly affected in virulence. These data highlight the importance of the high-affinity iron uptake system via an iron permease and a multicopper oxidase for biotrophic development in the U. maydis/maize (Zea mays) pathosystem. PMID:17138696

  1. The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain.

    Science.gov (United States)

    Ryndak, Michelle B; Wang, Shuishu; Smith, Issar; Rodriguez, G Marcela

    2010-02-01

    Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by Mycobacterium tuberculosis, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of M. tuberculosis in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in M. tuberculosis iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.

  2. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

    Science.gov (United States)

    Aweda, Tolulope A.; Meares, Claude F.

    2011-01-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (KA >108 M−1; KD calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate. PMID:21964396

  3. Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions.

    Science.gov (United States)

    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.

  4. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype

    DEFF Research Database (Denmark)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte

    2002-01-01

    with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable......Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor...... Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding...

  5. The occurrence and production of avidin: a new conception of the high-affinity biotin-binding protein.

    Science.gov (United States)

    Elo, H A; Korpela, J

    1984-01-01

    The production of avidin, a high-affinity biotin-binding egg-white protein, is not restricted to the avian, amphibian and reptilian oviducts. In the acute phase of inflammation, avidin is synthesized and secreted by various injured tissues in the domestic fowl, both male and female. Also in other avian species and a lizard, injured tissues produce an avidin-like biotin-binding factor. The non-oviductal production of avidin in domestic fowl has a great variety of inducers, for example acute inflammation caused by mechanical or thermal tissue injury, septic bacterial infection and (toxic) drugs, and even retrovirus-induced cell transformation. In culture, chicken embryo fibroblasts and yolk sac macrophages synthesize and secrete avidin. Besides the albumen, avidin may act as an antibacterial protein also in the tissues.

  6. Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

    Science.gov (United States)

    Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

    2014-05-01

    Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

  7. A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    ZHAO Lu; HE Cheng-yan; LIU Yan-li; ZHOU Hong-lan; ZHOU Jin-song; SHANG De-jing; YANG Hong

    2004-01-01

    A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(Kd=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.

  8. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R

    2005-01-01

    acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB...... analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported......Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites...

  9. Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

    Directory of Open Access Journals (Sweden)

    Guy Caljon

    Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

  10. High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction

    Energy Technology Data Exchange (ETDEWEB)

    Karatas, Hacer; Townsend, Elizabeth C; Cao, Fang; Chen, Yong; Bernard, Denzil; Liu, Liu; Lei, Ming; Dou, Yali; Wang, Shaomeng [Michigan; (HHMI)

    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.

  11. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

    Science.gov (United States)

    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.

  12. Cyr61/CCN1 displays high-affinity binding to the somatomedin B(1-44 domain of vitronectin.

    Directory of Open Access Journals (Sweden)

    Ivo M B Francischetti

    Full Text Available BACKGROUND: Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV family of extracellular-associated (matricellular proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP, von Willebrand factor type C (vWF, thrombospondin type 1 (TSP, and C-terminal growth factor cysteine knot (CT domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed. METHODS AND FINDINGS: In this report, surface plasmon resonance (SPR experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC with K(D in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB(1-44 of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin alphav beta3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB(1-44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB(1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB(1-44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, beta-endorphin, and other molecules. CONCLUSIONS: The finding that Cyr61 interacts with the SMTB(1-44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.

  13. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    Science.gov (United States)

    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.

  14. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

    Directory of Open Access Journals (Sweden)

    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  15. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

    Science.gov (United States)

    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

  16. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2016-10-01

    Full Text Available 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 (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith 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.

  17. Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. (Genentech, Inc., South San Francisco, CA (USA))

    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.

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

    Science.gov (United States)

    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.

  19. Viral reverse transcriptases show selective high affinity binding to DNA-DNA primer-templates that resemble the polypurine tract.

    Directory of Open Access Journals (Sweden)

    Gauri R Nair

    Full Text Available Previous results using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment-based approach that selected DNA primer-template duplexes binding with high affinity to HIV reverse transcriptase (RT showed that primers mimicking the 3' end, and in particular the six nt terminal G tract, of the RNA polypurine tract (PPT; HIV PPT: 5'-AAAAGAAAAGGGGGG-3' were preferentially selected. In this report, two viral (Moloney murine leukemia virus (MuLV and avian myeloblastosis virus (AMV and one retrotransposon (Ty3 RTs were used for selection. Like HIV RT, both viral RTs selected duplexes with primer strands mimicking the G tract at the PPT 3' end (AMV PPT: 5'-AGGGAGGGGGA-3'; MuLV PPT: 5'-AGAAAAAGGGGGG-3'. In contrast, Ty3, whose PPT lacks a G tract (5'-GAGAGAGAGGAA-3' showed no selective binding to any duplex sequences. Experiments were also conducted with DNA duplexes (termed DNA PPTs mimicking the RNA PPT-DNA duplex of each virus and a control duplex with a random DNA sequence. Retroviral RTs bound with high affinity to all viral DNA PPT constructs, with HIV and MuLV RTs showing comparable binding to the counterpart DNA PPT duplexes and reduced affinity to the AMV DNA PPT. AMV RT showed similar behavior with a modest preference for its own DNA PPT. Ty3 RT showed no preferential binding for its own or any other DNA PPT and viral RTs bound the Ty3 DNA PPT with relatively low affinity. In contrast, binding affinity of HIV RT to duplexes containing the HIV RNA PPT was less dependent on the G tract, which is known to be pivotal for efficient extension. We hypothesize that the G tract on the RNA PPT helps shift the binding orientation of RT to the 3' end of the PPT where extension can occur.

  20. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain.

    Science.gov (United States)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R; de Lichtenberg, Anne; Nielsen, Birgitte; Frølund, Bente; Brehm, Lotte; Clausen, Rasmus P; Bräuner-Osborne, Hans

    2005-10-01

    Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have synthesized and assayed three conformationally restricted GHB analogs for binding against the GHB-specific ligand [3H]NCS-382 [(E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-)acetic acid] in rat brain homogenate. The cyclohexene and cyclopentene analogs, 3-hydroxycyclohex-1-enecarboxylic acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported to date. Neither of the cycloalkenes showed any affinity (IC50 > 1 mM) for GABA(A) or GABA(B) receptors. These compounds show excellent potential as lead structures and novel tools for studying specific GHB receptor-mediated pharmacology.

  1. The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron

    NARCIS (Netherlands)

    Miethke, Marcus; Monteferrante, Carmine G.; Marahiel, Mohamed A.; van Dijl, Jan Maarten

    2013-01-01

    Efficient uptake of iron is of critical importance for growth and viability of microbial cells. Nevertheless, several mechanisms for iron uptake are not yet clearly defined. Here we report that the widely conserved transporter EfeUOB employs an unprecedented dual-mode mechanism for acquisition of

  2. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina

    2017-01-01

    the (11)C-labeling and subsequent evaluation of [(11)C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative......γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete...... understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report...

  3. Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

    Science.gov (United States)

    Kanomata, Naoki; Matsuura, Shiro; Nomura, Tsunehisa; Kurebayashi, Junichi; Mori, Taisuke; Kitawaki, Jo; Moriya, Takuya

    2017-01-01

    Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and

  4. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    Science.gov (United States)

    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.

  5. Novel fluo-4 analogs for fluorescent calcium measurements.

    Science.gov (United States)

    Martin, Vladimir V; Beierlein, Michael; Morgan, Josh L; Rothe, Anca; Gee, Kyle R

    2004-12-01

    We report new fluorescent calcium indicators based on fluo-4. Attachment of a carboxamide or methylenecarboxamide moiety to the BAPTA chelator portion of fluo-4 allowed for the attachment of dextrans, protein-reactive moieties, and biotin. In particular, a high affinity fluo-4 dextran conjugate was prepared and shown to be functional in brain slices. All new probes were characterized spectroscopically and exhibited large fluorescence increases upon calcium-binding. The biotinylated version of fluo-4 formed a persistent streptavidin complex which still responded to increasing calcium concentrations with a large fluorescence increase.

  6. Comparative uptake kinetics and transport of cadmium and phosphate in Phleum pratense-Glomus deserticolum associations

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, P.T.; Kapustka, L.A. (Miami Univ., Oxford, OH (United States))

    1993-01-01

    Mycorrhizal plants (timothy grass, Phleum pretense with Glomus deserticolum) were compared to nonmycorrhizal timothy grass to determine the effect of the mycorrhizal condition on the uptake and transport of cadmium. Companion experiments were conducted to ascertain phosphate uptake kinetics of mycorrhizal and nonmycorrhizal plants. Divalent cation competition experiments also were employed in this study. Comparisons of the high-affinity uptake mechanisms between mycorrhizal and nonmycorrhizal plants identified higher levels of phosphate uptake were due to an increase in the number of uptake sites rather than to differences in affinity. The respective values for K[sub m] for high-affinity phosphate uptake were 2.5 [plus minus] 1.3 [mu]MP (mycorrhizal) and 3.4 [plus minus] 1.3 [mu]MP (nonmycorrhizal), but these values were not statistically different at the [alpha] = 0.05 level. High-affinity Cd[sup 2+] uptake differed significantly between mycorrhizal (4.5 [plus minus] 2.8 [mu]M) and nonmycorrhizal (2.8 [plus minus] 1.1 [mu]M) plants. Presence of Ca[sup 2+] at 1.0mM concentration conferred considerable competitive protection in both the mycorrhizal and the nonmycorrhizal conditions. The effect of Ca[sup 2+] was an approximate fourfold increase in the respective K[sub m] values.

  7. SbHKT1;4, a member of the high-affinity potassium transporter gene family from Sorghum bicolor, functions to maintain optimal Na+/K+ balance under Na+ stress

    Institute of Scientific and Technical Information of China (English)

    Tian-Tian Wang; Zhi-Jie Ren; Zhi-Quan Liu; Xue Feng; Rui-Qi Guo; Bao-Guo Li; Le-Gong Li; HaiChun Jing

    2014-01-01

    In halophytic plants, the high-affinity potassium transporter HKT gene family can selectively uptake Kþ in the presence of toxic concentrations of Naþ. This has so far not been well examined in glycophytic crops. Here, we report the characterization of SbHKT1;4, a member of the HKT gene family from Sorghum bicolor. Upon Naþ stress, SbHKT1;4 expression was more strongly upregulated in salt-tolerant sorghum accession, correlating with a better balanced Naþ/Kþ ratio and enhanced plant growth. Heterogeneous expression analyses in mutants of Saccharomyces cerevisiae and Arabidopsis thaliana indicated that overexpressing SbHKT1;4 resulted in hypersensitivity to Naþ stress, and such hypersensitivity could be alleviated with the supply of elevated levels of Kþ, implicating that SbHKT1;4 may mediate Kþ uptake in the presence of excessive Naþ. Further electrophysiological evidence demonstrated that SbHKT1;4 could transport Naþ and Kþ when expressed in Xenopus laevis oocytes. The relevance of the finding that SbHKT1;4 functions to maintain optimal Naþ/Kþ balance under Naþ stress to the breeding of salt-tolerant glycophytic crops is discussed.

  8. Characteristics and Osmoregulatory Roles of Uptake Systems for Proline and Glycine Betaine in Lactococcus lactis

    NARCIS (Netherlands)

    Molenaar, Douwe; Hagting, Anja; Alkema, Harmen; Driessen, Arnold J.M.; Konings, Wilhelmus

    1993-01-01

    Lactococcus lactis subsp. lacti ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 µM is expressed constitutively. The

  9. Characteristics and Osmoregulatory Roles of Uptake Systems for Proline and Glycine Betaine in Lactococcus lactis

    NARCIS (Netherlands)

    Molenaar, Douwe; Hagting, Anja; Alkema, Harmen; Driessen, Arnold J.M.; Konings, Wilhelmus

    Lactococcus lactis subsp. lacti ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 µM is expressed constitutively. The

  10. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    Science.gov (United States)

    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.

  11. Fragile X mental retardation protein recognition of G quadruplex structure per se is sufficient for high affinity binding to RNA.

    Science.gov (United States)

    Bole, Medhavi; Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2008-12-01

    Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.

  12. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    Energy Technology Data Exchange (ETDEWEB)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. (Centre de Biochimie, Nice (France))

    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.

  13. G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

    Science.gov (United States)

    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

  14. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

    Directory of Open Access Journals (Sweden)

    Maria José Leandro

    Full Text Available 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.

  15. High affinity melatonin receptors in the vertebrate brain: implications for the control of the endogenous oscillatory systems.

    Science.gov (United States)

    Fraschini, F; Stankov, B

    1994-01-01

    Currently, the melatonin receptor is depicted as a membrane-associated protein, linked to a guanine nucleotide-binding protein (G-protein), and thus the melatonin receptor represents a member of a receptor superfamily, acting through G-proteins in the first step of their signal-transduction pathways. Although on a number of occasions specific binding of radioactive melatonin has been demonstrated in a wide variety of tissues and organs, to date, high affinity G-protein-regulated melatonin binding sites, suggestive for a functional melatonin receptor, have been convincingly confirmed in the brain only. There is a significant species variation in the distribution of the melatonin receptor in the vertebrate brain. The limited number of studies prevents any definitive conclusion in terms of phylogeny, though generally speaking, the lower vertebrates' brains tend to express melatonin receptors with wider distribution. Two sites have been consistently found to express high density of melatonin receptors: the pars tuberalis of the adenohypophysis and the hypothalamic suprachiasmatic nuclei (SCN). It must be pointed out, however, that there are some exceptions. Binding in the human pars tuberalis has not been reported, and apparently, the sheep and the mustelids' suprachiasmatic nuclei do not express detectable binding. The function of melatonin in pars tuberalis is unclear, and the control of the synthesis (and release) of paracrine factors that act at site(s) distant from the melatonin target cells, have been suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    Science.gov (United States)

    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.

  17. High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes.

    Science.gov (United States)

    Lang, Benjamin; Blot, Nicolas; Bouffartigues, Emeline; Buckle, Malcolm; Geertz, Marcel; Gualerzi, Claudio O; Mavathur, Ramesh; Muskhelishvili, Georgi; Pon, Cynthia L; Rimsky, Sylvie; Stella, Stefano; Babu, M Madan; Travers, Andrew

    2007-01-01

    The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

  18. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  20. A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

    Directory of Open Access Journals (Sweden)

    Eleonora Dehlink

    Full Text Available 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.

  1. The high affinity IgE receptor (FcεRI) expression and function in airway smooth muscle.

    Science.gov (United States)

    Redhu, Naresh Singh; Gounni, Abdelilah S

    2013-02-01

    The airway smooth muscle (ASM) is no longer considered as merely a contractile apparatus and passive recipient of growth factors, neurotransmitters and inflammatory mediators signal but a critical player in the perpetuation and modulation of airway inflammation and remodeling. In recent years, a molecular link between ASM and IgE has been established through Fc epsilon receptors (FcεRs) in modulating the phenotype and function of these cells. Particularly, the expression of high affinity IgE receptor (FcεRI) has been noted in primary human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthmatic subjects. The activation of FcεRI on ASM cells suggests a critical yet almost completely ignored network which may modulate ASM cell function in allergic asthma. This review is intended to provide a historical perspective of IgE effects on ASM and highlights the recent updates in the expression and function of FcεRI, and to present future perspectives of activation of this pathway in ASM cells.

  2. The High Affinity IgE Receptor (FcεRI as a Target for Anti-allergic Agents

    Directory of Open Access Journals (Sweden)

    Kyoko Takahashi

    2005-01-01

    Full Text Available Prevention of the effector cell activation via high affinity IgE receptor (FcεRI is thought to be a straightforward strategy for suppressing the allergic reaction. Among the numerous methods to prevent the activation through FcεRI, three versions are described in this article. The first and second ideas involve inhibition of binding between FcεRI and IgE with a soluble form of the FceRI α chain and a humanized antibody directed against the a chain, respectively. Both of these paths involve suppression the histamine release from human peripheral blood basophils in vitro. They also inhibited the allergic reaction in vivo. The soluble α inhibited the anaphylactic reaction in rodents and the Fab fragments of the humanized anti-FcεRI α chain antibody suppressed the dermal response in rhesus monkeys. The third idea involves repression of FcεRI expression by suppressing the transcription of the genes encoding the subunits of FceRI. Although no plausible candidate molecule for actualizing this idea can be identified at present, further analyses of the transcriptional regulatory mechanisms in the human FcεRI α and β chain genes will lead to the discovery of novel targets for developing anti-allergic agents.

  3. ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  5. Synthesis of tetravalent LacNAc-glycoclusters as high-affinity cross-linker against Erythrina cristagalli agglutinin.

    Science.gov (United States)

    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.

  6. A Class of High-affinity Bicyclooctane G551D-CFTR Activators Identified by High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    HE Cheng-yan; ZHAO Lu; LIU Yan-li; XU Li-na; SHANG De-jing; YANG Hong

    2004-01-01

    The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.

  7. VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Barelle

    2015-09-01

    Full Text Available At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

  8. ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

    Directory of Open Access Journals (Sweden)

    Anuradha Kuchibhatla

    Full Text Available A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0 pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

  9. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion (Cornell); (UAB); (Florida)

    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.

  10. Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1)

    National Research Council Canada - National Science Library

    Wanichawan, Pimthanya; Hodne, Kjetil; Hafver, Tandekile Lubelwana; Lunde, Marianne; Martinsen, Marita; Louch, William Edward; Sejersted, Ole Mathias; Carlson, Cathrine Rein

    2016-01-01

    NCX1 (Na(+)/Ca(2+) exchanger 1) is an important regulator of intracellular Ca(2+) and a potential therapeutic target for brain ischaemia and for diastolic heart failure with preserved ejection fraction. PLM (phospholemman...

  11. Taking advantage: high-affinity B cells in the germinal center have lower death rates, but similar rates of division, compared to low-affinity cells.

    Science.gov (United States)

    Anderson, Shannon M; Khalil, Ashraf; Uduman, Mohamed; Hershberg, Uri; Louzoun, Yoram; Haberman, Ann M; Kleinstein, Steven H; Shlomchik, Mark J

    2009-12-01

    B lymphocytes producing high-affinity Abs are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high-affinity B cells are selected during the immune response has never been elucidated. Although it has been shown that high-affinity cells directly outcompete low-affinity cells in the germinal center (GC), whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity cells proliferate more rapidly or are more likely to enter cell cycle, thereby outgrowing lower affinity cells. Alternatively, higher affinity cells could be relatively more resistant to cell death in the GC. By comparing high- and low-affinity B cells for the same Ag, we show here that low-affinity cells have an intrinsically higher death rate than do cells of higher affinity, even in the absence of competition. This suggests that selection in the GC reaction is due at least in part to the control of survival of higher affinity B cells and not by a proliferative advantage conferred upon these cells compared with lower affinity B cells. Control over survival rather than proliferation of low- and high-affinity B cells in the GC allows greater diversity not only in the primary response but also in the memory response.

  12. Replacement of the Bryostatin A- and B-Pyran Rings With Phenyl Rings Leads to Loss of High Affinity Binding With PKC.

    Science.gov (United States)

    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'.

  13. High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4(+) T cell responses more than 30 years post-vaccinia virus vaccination

    DEFF Research Database (Denmark)

    Wang, M.; Tang, Sheila Tuyet; Lund, Ole;

    2009-01-01

    Interferon-gamma secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human...

  14. High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila

    NARCIS (Netherlands)

    Ramírez, Fidel; Lingg, Thomas; Toscano, Sarah; Lam, Kin Chung; Georgiev, Plamen; Chung, Ho-Ryun; Lajoie, Bryan R; de Wit, Elzo; Zhan, Ye; de Laat, Wouter; Dekker, Job; Manke, Thomas; Akhtar, Asifa

    2015-01-01

    Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific le

  15. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.;

    2006-01-01

    as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile...

  16. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

    Science.gov (United States)

    Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

    2001-01-01

    BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

  17. A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

    DEFF Research Database (Denmark)

    Bach, Anders*; Clausen, Bettina H; Møller, Magda;

    2012-01-01

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors...

  18. Rhodamine-labeled 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane analogues as high-affinity fluorescent probes for the dopamine transporter

    DEFF Research Database (Denmark)

    Cha, Joo Hwan; Zou, Mu-Fa; Adkins, Erika M

    2005-01-01

    Novel fluorescent ligands were synthesized to identify a high-affinity probe that would enable visualization of the dopamine transporter (DAT) in living cells. Fluorescent tags were extended from the N- or 2-position of 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane, using an ethylamino lin...

  19. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

    Science.gov (United States)

    Levay, Agata; Brenneman, Randall; Hoinka, Jan; Sant, David; Cardone, Marco; Trinchieri, Giorgio; Przytycka, Teresa M; Berezhnoy, Alexey

    2015-07-13

    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.

  20. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding

    Science.gov (United States)

    Avery, Adam W.; Crain, Jonathan; Thomas, David D.; Hays, Thomas S.

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  1. Gonyautoxin 1/4 aptamers with high-affinity and high-specificity: From efficient selection to aptasensor application.

    Science.gov (United States)

    Gao, Shunxiang; Hu, Bo; Zheng, Xin; Cao, Ying; Liu, Dejing; Sun, Mingjuan; Jiao, Binghua; Wang, Lianghua

    2016-05-15

    Gonyautoxin 1/4 (GTX1/4) are potent marine neurotoxins with significant public health impact. However, the ethical issues and technical defects associated with the currently applied detection methods for paralytic shellfish toxin GTX1/4 are pressing further studies to develop suitable alternatives in a regulatory monitoring system. This work describes the first successful selection, optimization, and characterization of an aptamer that bind with high affinity and specificity to GTX1/4. Compared to the typical MB-SELEX, GO-SELEX, an advanced screening technology, has significant advantages for small molecular aptamer development. Furthermore, we truncated GTX1/4 aptamer and obtained the aptamer core sequence with a higher Kd of 17.7 nM. The aptamer GO18-T-d was then used to construct a label-free and real-time optical BLI aptasensor for the detection of GTX1/4. The aptasensor showed a broad detection range from 0.2 to 200 ng/mL GTX1/4 (linear range from 0.2 to 90 ng/mL), with a low detection limit of 50 pg/mL. Moreover, the aptasensor exhibited a high degree of specificity for GTX1/4 and no cross reactivity to other marine toxins. The aptasensor was then applied to the detection of GTX1/4 in spiked shellfish samples and showed a good reproducibility and stability. We believe that this novel aptasensor offers a promising alternative to traditional analytical methods for the rapid detection of the marine biotoxin GTX1/4. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Plant High-Affinity Potassium (HKT Transporters Involved in Salinity Tolerance: Structural Insights to Probe Differences in Ion Selectivity

    Directory of Open Access Journals (Sweden)

    Maria Hrmova

    2013-04-01

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

    Science.gov (United States)

    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.

  4. Effect of repeated nicotine exposure on high-affinity nicotinic acetylcholine receptor density in spontaneously hypertensive rats.

    Science.gov (United States)

    Hohnadel, Elizabeth J; Hernandez, Caterina M; Gearhart, Debra A; Terry, Alvin V

    Spontaneously hypertensive rats (SHRs) are often used as a model of attention deficit hyperactivity disorder (ADHD) and to investigate the effects of hypertension on cognitive function. Further, they appear to have reduced numbers of central nicotinic acetylcholine receptors (nAChRs) and, therefore, may be useful to model certain aspects of Alzheimer's disease (AD) and other forms of dementia given that a decrease in nAChRs is thought to contribute to cognitive decline in these disorders. In the present study, based on reports that chronic nicotine exposure increases nAChRs in several mammalian models, we tested the hypothesis that repeated exposures to a relatively low dose of the alkaloid would ameliorate the receptor deficits in SHR. Thus, young-adult SHRs and age-matched Wistar-Kyoto (WKY) control rats were treated with either saline or nicotine twice a day for 14 days (total daily dose = 0.7 mg/kg nicotine base) and then sacrificed. Quantitative receptor autoradiography with [125I]-IPH, an epibatidine analog, revealed: (1) that high-affinity nAChRs were higher in saline-treated WKY (control) rats compared to saline-treated SHRs in 18 of the 19 brain region measured, although statistically different only in the mediodorsal thalamic nuclei, (2) that nicotine significantly increased nAChR binding in WKY rats in six brain areas including cortical regions and the anterior thalamic nucleus, (3) that there were no cases where nicotine significantly increased nAChR binding in SHRs. These results indicate that subjects deficient in nAChRs may be less sensitive to nAChR upregulation with nicotine than normal subjects and require higher doses or longer periods of exposure.

  5. Fast-onset lidocaine block of rat NaV1.4 channels suggests involvement of a second high-affinity open state.

    Science.gov (United States)

    Gingrich, Kevin J; Wagner, Larry E

    2016-06-01

    Local anesthetics (LAs) block resting, open, and inactivated states of voltage-gated Na(+) channels where inactivated states are thought to bind with highest affinity. However, reports of fast-onset block occurring over milliseconds hint at high-affinity block of open channels. Movement of voltage-sensor domain IV-segment 4 (DIVS4) has been associated with high affinity LA block termed voltage-sensor block (VSB) that also leads to a second open state. These observations point to a second high-affinity open state that may underlie fast-onset block. To test for this state, we analyzed the modulation of Na(+) currents by lidocaine and its quaternary derivative (QX222) from heterologously expressed (Xenopus laevis oocytes) rat skeletal muscle μ1 NaV1.4 (rSkM1) with β1 (WT-β1), and a mutant form (IFM-QQQ mutation in the III-IV interdomain, QQQ) lacking fast inactivation, in combination with Markov kinetic gating models. 100 μM lidocaine induced fast-onset (τonset≈2 ms), long-lived (τrecovery≈120 ms) block of WT-β1 macroscopic currents. Lidocaine blocked single-channel and macroscopic QQQ currents in agreement with our previously described mechanism of dual, open-channel block (DOB mechanism). A DOB kinetic model reproduced lidocaine effects on QQQ currents. The DOB model was extended to include trapping fast-inactivation and activation gates, and a second open state (OS2); the latter arising from DIVS4 translocation that precedes inactivation and exhibits high-affinity, lidocaine binding (apparent Kd=25 μM) that accords with VSB (DOB-S2VSB mechanism). The DOB-S2VSB kinetic model predicted fast-onset block of WT-β1. The findings support the involvement of a second, high-affinity, open state in lidocaine modulation of Na(+) channels.

  6. Calcium in plant cells

    Directory of Open Access Journals (Sweden)

    V. V. Schwartau

    2014-04-01

    Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you

  7. Uptake of gamma-aminobutyric acid and L-glutamic acid by synaptosomes from postmortem human cerebral cortex: multiple sites, sodium dependence and effect of tissue preparation.

    Science.gov (United States)

    Dodd, P R; Watson, W E; Morrison, M M; Johnston, G A; Bird, E D; Cowburn, R F; Hardy, J A

    1989-06-26

    The uptake of gamma-aminobutyric acid (GABA) and L-glutamic acid by synaptosomes prepared from frozen postmortem human brain was shown to be effected via distinct high and low affinity sites. At approximately 17 h postmortem delay, the kinetic parameters for GABA uptake were: high affinity site, Km 7.1 +/- 2.5 microM, Vmax 18.7 +/- 4.8 nmol.min-1 per 100 mg protein; low affinity site, Km 2 +/- 1 mM, Vmax 425 +/- 250 nmol.min-1 per 100 mg protein (means +/- S.E.M., n = 13). Kinetic parameters for L-glutamate uptake were: high affinity site, Km 7.5 +/- 1.0 microM, Vmax 85 +/- 8 nmol.min-1 per 100 mg protein; low affinity site, Km 1.8 +/- 1.2 mM. Vmax 780 +/- 175 nmol.min-1 per 100 mg protein (n = 11). A detailed kinetic analysis of high affinity GABA uptake was performed over a range of sodium ion concentrations. The results were consistent with a coupling ratio of one Na+ ion to one GABA molecule; a similar result was found with rat brain synaptosomes. However, rat and human synaptosomes differed in the degree to which the substrate affinity of the high affinity GABA uptake site varied with decreasing Na+ ion concentration. High affinity GABA uptake was markedly affected by the method used to freeze and divide the tissue, but did not vary greatly in different cortical regions. There was some decline of high affinity GABA uptake activity with postmortem delay, apparently due to a loss of sites rather than a change in site affinity.

  8. The sarcoplasmic calcium pump - a most efficient ion translocating system.

    Science.gov (United States)

    Hasselbach, W

    1977-04-21

    In contrast to the sodium-potassium transporting plasma membranes, the sarcoplasmic membranes (SR) are highly specialized structures into which only two major intrinsic proteins, a calcium transporting protein and a calcium binding protein are embedded. The calcium transporting protein is a highly asymmetric molecule. It binds two calcium ions with a very high affinity at its external, and two calcium ions with low affinity at the internal section of the molecule. ATP is bound with high afffinity to an external binding site, inducing a conformational change. When the vesicular membranes are exposed to solutions containing Ca++, Mg++ and ATP, ATP is hydrolyzed and simultaneously calcium ions are translocated from the external medium into the vesicular space. When calcium ions are translocated in the opposite direction, ATP is synthesized. The calcium-ATP ratio for ATP cleavage as well as for ATP synthesis is 2. Thus, the SR membranes can transform reversibly chemical into osmotical energy. Inward and outward movements of calcium ions are relatively slow processes connected with the appearance and disappearance of different phosphorylated intermediates. One phosphorylated intermediate is formed by phosphoryltransfer from ATP when calcium ions are present in the medium. In contrast, when calcium ions are absent from the external medium, two different intermediates can be formed by the incorporation of inorganic phosphate. Only when calcium ions present in the internal space of the vesicles are released, the incorporation of inorganic phosphate gives rise to an intermediate who phosphoryl group can be transferred to ADP.

  9. The use of size-defined DNA-functionalized calcium phosphate nanoparticles to minimise intracellular calcium disturbance during transfection.

    Science.gov (United States)

    Neumann, Sebastian; Kovtun, Anna; Dietzel, Irmgard D; Epple, Matthias; Heumann, Rolf

    2009-12-01

    Calcium phosphate-based transfection methods are frequently used to transfer DNA into living cells. However, it has so far not been studied in detail to what extend the different transfection methods lead to a net calcium uptake. Upon subsequent resolution of the calcium phosphate, intracellular free ionic calcium-surges could result, inducing as side effect various physiological responses that may finally result in cell death. Here we investigated the overall calcium uptake by the human bladder carcinoma cell line T24 during the standard calcium phosphate transfection method and also during transfection with custom-made calcium phosphate/DNA nanoparticles by isotope labelling with (45)calcium. (45)Calcium uptake was strongly increased after 7h of standard calcium phosphate transfection but not if the transfection was performed with calcium phosphate nanoparticles. Time lapse imaging microscopy using the calcium-sensitive dye Fura-2 revealed large transient increases of the intracellular free calcium level during the standard calcium phosphate transfection but not if calcium phosphate nanoparticles were used. Consistently, the viability of cells transfected by calcium phosphate/DNA nanoparticles was not changed, in remarkable contrast to the standard method where considerable cell death occurred.

  10. Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

  11. BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction.

    Science.gov (United States)

    Uegaki, Koichi; Kumanogoh, Haruko; Mizui, Toshiyuki; Hirokawa, Takatsugu; Ishikawa, Yasuyuki; Kojima, Masami

    2017-05-12

    Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function

  12. Human Eosinophils Express the High Affinity IgE Receptor, FcεRI, in Bullous Pemphigoid

    Science.gov (United States)

    Messingham, Kelly N.; Holahan, Heather M.; Frydman, Alexandra S.; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A.

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  13. Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

    Directory of Open Access Journals (Sweden)

    Kelly N Messingham

    Full Text Available Bullous pemphigoid (BP is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen. Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1 To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2 To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16 with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils.

  14. Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

    Science.gov (United States)

    Tiago, Teresa; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2004-05-11

    Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.

  15. A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids.

    Science.gov (United States)

    Ieong, Ka-Weng; Pavlov, Michael Y; Kwiatkowski, Marek; Ehrenberg, Måns; Forster, Anthony C

    2014-05-01

    There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA(PheB) body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA(AlaB) body than from the tRNA(PheB) body. At ∼1 µM EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNA(PheB), especially in the case of the bulky bK. In contrast, the swap to the tRNA(AlaB) body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA(AlaB) and tRNA(PheB) bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.

  16. Synthesis and pre-clinical evaluation of a new class of high-affinity {sup 18}F-labeled PSMA ligands for detection of prostate cancer by PET imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, James; Amor-Coarasa, Alejandro; Williams, Clarence; Ponnala, Shashikanth [Weill Cornell Medicine, Division of Radiopharmaceutical Sciences and Molecular Imaging Innovations Institute, Department of Radiology, New York, NY (United States); Nikolopoulou, Anastasia [Weill Cornell Medicine, Division of Radiopharmaceutical Sciences and Molecular Imaging Innovations Institute, Department of Radiology, New York, NY (United States); Weill Cornell Medicine, Citigroup Biomedical Imaging Center, New York, NY (United States); Kim, Dohyun [Weill Cornell Medicine, Citigroup Biomedical Imaging Center, New York, NY (United States); Babich, John W. [Weill Cornell Medicine, Division of Radiopharmaceutical Sciences and Molecular Imaging Innovations Institute, Department of Radiology, New York, NY (United States); Weill Cornell Medicine, Citigroup Biomedical Imaging Center, New York, NY (United States); Weill Cornell Medicine, Meyer Cancer Center, New York, NY (United States)

    2017-04-15

    Current clinical imaging of PSMA-positive prostate cancer by positron emission tomography (PET) mainly features {sup 68}Ga-labeled tracers, notably [{sup 68}Ga]Ga-PSMA-HBED-CC. The longer half-life of fluorine-18 offers significant advantages over Ga-68, clinically and logistically. We aimed to develop high-affinity PSMA inhibitors labeled with fluorine-18 as alternative tracers for prostate cancer. Six triazolylphenyl ureas and their alkyne precursors were synthesized from the Glu-urea-Lys PSMA binding moiety. PSMA affinity was determined in a competitive binding assay using LNCaP cells. The [{sup 18}F]triazoles were isolated following a Cu(I)-catalyzed click reaction between the alkynes and [{sup 18}F]fluoroethylazide. The {sup 18}F-labeled compounds were evaluated in nude mice bearing LNCaP tumors and compared to [{sup 68}Ga]Ga-PSMA-HBED-CC and [{sup 18}F]DCFPyL. Biodistribution studies of the two tracers with the highest imaged-derived tumor uptake and highest PSMA affinity were undertaken at 1 h, 2 h and 4 h post-injection (p.i.), and co-administration of PMPA was used to determine whether uptake was PSMA-specific. F-18-labeled triazolylphenyl ureas were prepared with a decay-corrected RCY of 20-40 %, >98 % radiochemical and chemical purity, and specific activity of up to 391 GBq/μmol. PSMA binding (IC{sub 50}) ranged from 3-36 nM. The position of the triazole influenced tumor uptake (3 > 4 > 2), and direct conjugation of the triazole with the phenylurea moiety was preferred to insertion of a spacer group. Image-derived tumor uptake ranged from 6-14 %ID/g at 2 h p.i., the time of maximum tumor uptake; uptake of [{sup 68}Ga]Ga-PSMA-HBED-CC and [{sup 18}F]DCFPyL was 5-6 %ID/g at 1-3 h p.i., the time of maximum tumor uptake. Biodistribution studies of the two most promising compounds gave maximum tumor uptakes of 10.9 ± 1.0 % and 14.3 ± 2.5 %ID/g, respectively, as compared to 6.27 ± 1.44 %ID/g for [{sup 68}Ga]Ga-PSMA-HBED-CC. Six [{sup 18}F

  17. High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.

    Science.gov (United States)

    Schalch, Thomas; Job, Godwin; Noffsinger, Victoria J; Shanker, Sreenath; Kuscu, Canan; Joshua-Tor, Leemor; Partridge, Janet F

    2009-04-10

    In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

  18. N- and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor

    DEFF Research Database (Denmark)

    Hansen, L S; Sparre-Ulrich, A H; Christensen, M.

    2016-01-01

    BACKGROUND AND PURPOSE: Glucose-dependent insulinotropic polypeptide (GIP) impacts lipid, bone, and glucose homeostasis. The GIP receptor belongs to G protein-coupled receptor family B1 and signals through GαS. High affinity ligands for in vivo use are needed to elucidate GIP's physiological...... functions and pharmacological potential. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here we characterize eight N-terminal trrncations of human GIP(1-30)NH2 : GIP(2- to 9-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with the human GIP receptor and assessed......, but superior antagonist GIP(3-30)NH2 , that together with GIP(5-30)NH2 were high-affinity competitive antagonist and thus may be suitable tool compounds for basic GIP research and future pharmacological interventions....

  19. A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra-high affinity ABC system distinct from the molybdate transporter.

    Science.gov (United States)

    Smart, Jonathan P; Cliff, Matthew J; Kelly, David J

    2009-11-01

    The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium.

  20. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    Science.gov (United States)

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

  1. Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.

    Science.gov (United States)

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A; Lowe, Edward D; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W; Cogdell, Richard J; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-08-28

    How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups.

  2. Structures of the Ultra-High-Affinity Protein–Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa

    Science.gov (United States)

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A.; Lowe, Edward D.; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W.; Cogdell, Richard J.; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups. PMID:26215615

  3. Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

    Science.gov (United States)

    Brophy, Megan Brunjes; Nakashige, Toshiki G; Gaillard, Aleth; Nolan, Elizabeth M

    2013-11-27

    Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96-114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn(II) coordination sphere in solution, affording an unprecedented biological His6 site. These solution studies are in agreement with a Mn(II)-CP crystal structure reported recently (Damo, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3841). Remarkably high-affinity Mn(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103-105 to 104-106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against Escherichia coli and Staphylococcus aureus. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn(II) binding; the S100A7, S100A9(C3S), S100A12, and S100B homodimers do not exhibit such Mn(II)-binding capacity.

  4. The levels of boron-uptake proteins in roots are correlated with tolerance to boron stress in barley

    Science.gov (United States)

    Boron (B) is an essential micronutrient required for plant growth and development. Recently, two major B-uptake proteins, BOR1 and NIP5;1 have been identified and partially characterized. BOR1 is a high-affinity B transporter involved in xylem loading in roots, and NIP5;1 acts is a major boric-acid ...

  5. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

    Science.gov (United States)

    Burel, Sebastien A; Hart, Christopher E; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-Hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M; Hung, Gene; Dan, Amy; Prakash, T P; Seth, Punit P; Swayze, Eric E; Bennett, C Frank; Crooke, Stanley T; Henry, Scott P

    2016-03-18

    High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    Science.gov (United States)

    Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  7. Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes.

    Science.gov (United States)

    Kumar, Sunil; Xue, Liang; Arya, Dev P

    2011-05-18

    A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ∼10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT

  8. In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide receptor.

    Science.gov (United States)

    Ma, Lixin; Yu, Ping; Veerendra, Bhadrasetty; Rold, Tammy L; Retzloff, Lauren; Prasanphanich, Adam; Sieckman, Gary; Hoffman, Timothy J; Volkert, Wynn A; Smith, Charles J

    2007-01-01

    Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

  9. In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH2 Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor

    Directory of Open Access Journals (Sweden)

    Lixin Ma

    2007-05-01

    Full Text Available Gastrin-releasing peptide (GRP receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN, a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H2N-glycylglycylglycine-BBN[7–14]NH2 peptide with the following general sequence: H2N-G-G-G-Q-W-A-V-G-H-L-M-(NH2. This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H2N-G-G-G-BBN[7–14]NH2 in dimethylformamide (DMF. In vitro competitive binding assays, using 125I-Tyr4-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

  10. Computational comparison of a calcium-dependent jellyfish protein (apoaequorin) and calmodulin-cholesterol in short-term memory maintenance.

    Science.gov (United States)

    Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K

    2017-03-06

    Memory reconsolidation and maintenance depend on calcium channels and on calcium/calmodulin-dependent kinases regulating protein turnover in the hippocampus. Ingestion of a jellyfish protein, apoaequorin, reportedly protects and/or improves verbal learning in adults and is currently widely advertised for use by the elderly. Apoaequorin is a member of the EF-hand calcium binding family of proteins that includes calmodulin. Calmodulin-1 (148 residues) differs from Apoaequorin (195 residues) in that it contains four rather than three Ca(2+)-binding sites and three rather than four cholesterol-binding (CRAC, CARC) domains. All three cholesterol-binding CARC domains in calmodulin have a high interaction affinity for cholesterol compared to only two high affinity CARC domains in apoaequorin. Both calmodulin and apoaequorin can form dimers with a potential of eight bound Ca(2+) ions and six high affinity-bound cholesterol molecules in calmodulin with six bound Ca(2+) ions and a mixed population of eight cholesterols bound to both CARC and CRAC domains in apoaqueorin. MEMSAT-SVM analysis indicates that both calmodulin and apoaqueorin have a pore-lining region. The Peptide-Cutter algorithm predicts that calmodulin-1 contains 11 trypsin-specific cleavage sites (compared to 21 in apoaqueorin), four of which are potentially blocked by cholesterol and three are within the Ca-binding domains and/or the pore-lining region. Three are clustered between the third and fourth Ca(2+)-binding sites. Only calmodulin pore-lining regions contain Ca(2+) binding sites and as dimers may insert into the plasma membrane of neural cells and act as Ca(2+) channels. In a dietary supplement, bound cholesterol may protect both apoaequorin and calmodulin from proteolysis in the gut as well as facilitate uptake across the blood-brain barrier. Our results suggest that a physiological calmodulin-cholesterol complex, not cholesterol-free jellyfish protein, may better serve as a dietary supplement to

  11. Etiology of amyloidosis determines myocardial 99mTc-DPD uptake in amyloidotic cardiomyopathy.

    Science.gov (United States)

    Longhi, Simone; Bonfiglioli, Rachele; Obici, Laura; Gagliardi, Christian; Milandri, Agnese; Lorenzini, Massimiliano; Guidalotti, Pier Luigi; Merlini, Giampaolo; Rapezzi, Claudio

    2015-05-01

    Tc-DPD (Tc-3,3-diphosphono-1,2-propanodicarboxylic acid) has a high affinity for transthyretin (TTR)-infiltrated myocardium, allowing a differential diagnosis with light chain cardiac amyloidosis and other nonamyloidotic cardiomyopathies with a hypertrophic phenotype, in which myocardial tracer uptake is low or absent. Myocardial bone tracer uptake in the rarer forms of amyloidosis (eg, apolipoprotein-related) has been rarely studied. We present 4 cases of cardiac amyloidosis that underwent Tc-DPD scintigraphy; myocardial DPD uptake was present in patients with ATTR, wtTTR and apolipoprotein AI and negative in cases with AL and apolipoprotein AII-related disease.

  12. Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

    Institute of Scientific and Technical Information of China (English)

    LIANG; Tiebing

    2001-01-01

    ., 1996, 255: 373-386.[13]Kim, J. -S., Pabo, C. O., Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants, Proc. Natl. Acad. Sci. USA, 1998, 95: 2812-2817.[14]Wu, H., Yang, W. -P., BarbasIII, C. F., Building zinc fingers by selection: toward a therapeutic application, Proc. Natl. Acad. Sci. USA, 1995, 92: 344-348.[15]Wang, B. S., Pabo, C. O., Dimerization of zinc fingers mediated by peptides evolved in vitro from random sequences, Proc. Natl. Acad. Sci. USA, 1999, 96: 9568-9573.[16]Choo, Y., Sánchez-García, I., Klug, A., In vivo repression by a site-specific DNA-binding protein designed against an on-cogenic sequence, Nature, 1994, 372: 642-645.[17]Wolfe, S. A., Greisman, H. A., Ramm, E. I. et al., Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code, J. Mol. Biol., 1999, 285: 1917-1934.[18]Chen, J. -Q., Pongor, S., Simoncsits, A., Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs, Nucleic Acids Research, 1997, 25: 2047-2054.[19]Simoncsits, A., Tj?rnhammar, M. -L., Wang, S. -L. et al., Isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric DNA sequences containing the TTAA and TTAC subsites, Nucleic Acids Research, 1999, 27: 3474-3480.[20]Zhou, Y. -H., Busby, S., Ebright, R. H., Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers, Cell, 1993, 73: 375-379.[21]Studier, F. W., Rosenberg, A. H., Dunn, J. J. et al., Use of T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol., 1990, 185: 60-89.[22]Simoncsits, A., Bristulf, J., Tj?rnhammar, M. -L. et al., Deletion mutants of human interleukin 1? with significantly re-duced agonist properties: search for agonist/ antagonist switch in ligands to the interleukin 1

  13. Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Z.; Reiske, H.R.; Wilson, D.B.

    1999-11-01

    Two different Cd{sup 2+} uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn{sup 2+} uptake system which also takes up Cd{sup 2+} and is induced by Mn{sup 2+} starvation. The calculated K{sub m} and V{sub max} are 0.26 {mu}M and 3.6 {mu}mol g of dry cell{sup {minus}1} min{sup {minus}1}, respectively. Unlike Mn{sup 2+} uptake, which is facilitated by citrate and related tricarboxylic acids, Cd{sup 2+} uptake is weakly inhibited by citrate. Cd{sup 2+} and Mn{sup 2+} are competitive inhibitors of each other, and the affinity of the system for Cd{sup 2+} is higher than that for Mn{sup 2+}. The other Cd{sup 2+} uptake system is expressed in Mn{sup 2+}-sufficient cells, and no K{sub m} can be calculated for it because uptake is nonsaturable. Mn{sup 2+} does not compete for transport through this system, nor does any other tested cation, i.e., Zn{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Mg{sup 2+}, Ca{sup 2+}, Fe{sup 2+}, or Ni{sup 2+}. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn{sup 2+}-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn{sup 2+} for growth as the parental strain. Mn{sup 2+} starvation-induced Cd{sup 2+} uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn{sup 2+} or Cd{sup 2+} accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system.

  14. biodegradation and moisture uptake modified starch-filled linear low ...

    African Journals Online (AJOL)

    CHEMISTRY

    Key words: Starch-LLDPE blends, Calcium chloride, D-glucose, Chloroform and Alumina. INTRODUCTION .... rate of diffusion into the composites this is because moisture uptakes in ... prepared with oxidized potato starch. Food engineering ...

  15. Thyroid Scan and Uptake

    Science.gov (United States)

    ... News Physician Resources Professions Site Index A-Z Thyroid Scan and Uptake Thyroid scan and uptake uses ... the Thyroid Scan and Uptake? What is a Thyroid Scan and Uptake? A thyroid scan is a ...

  16. Thyroid Scan and Uptake

    Medline Plus

    Full Text Available ... News Physician Resources Professions Site Index A-Z Thyroid Scan and Uptake Thyroid scan and uptake uses ... the Thyroid Scan and Uptake? What is a Thyroid Scan and Uptake? A thyroid scan is a ...

  17. Thyroid Scan and Uptake

    Science.gov (United States)

    ... Physician Resources Professions Site Index A-Z Thyroid Scan and Uptake Thyroid scan and uptake uses small ... Thyroid Scan and Uptake? What is a Thyroid Scan and Uptake? A thyroid scan is a type ...

  18. A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Ramon Wahl

    2010-02-01

    Full Text Available 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.

  19. [Calcium and vitamin D in osteology].

    Science.gov (United States)

    Amling, M; Barvencik, F

    2015-06-01

    Calcium homeostasis is of paramount physiological and pathophysiological importance in health and disease. This article focuses on the skeletal relevance of calcium and vitamin D in daily clinical practice. Against the background of an endemic vitamin D deficiency in Germany and the increasing number of patients with drug-induced (proton pump inhibitor) enteral calcium uptake problems, it is of critical importance to understand that a vitamin D level of > 30 µg/l (> 75 nmol/l) is required for intact skeletal mineralization and that furthermore, a physiological gastric acid production is essential for a normal enteral uptake of calcium from foodstuffs. Therefore, a guideline-conform handling of vitamin D and calcium substitution is required not only for patients with rheumatoid diseases but also for any osteological therapy.

  20. High-affinity binding of southern African HIV type 1 subtype C envelope protein, gp120, to the CCR5 coreceptor.

    Science.gov (United States)

    Fromme, Bernhard J; Coetsee, Marla; Van Der Watt, Pauline; Chan, Mei-Chi; Sperling, Karin M; Katz, Arieh A; Flanagan, Colleen A

    2008-12-01

    HIV-1 subtype C is the fastest spreading subtype worldwide and predominantly uses the CCR5 coreceptor, showing minimal transition to the X4 phenotype. This raises the possibility that envelope proteins of HIV-1 subtype C have structural features that favor interaction with CCR5. Preference for CCR5 could arise from enhanced affinity of HIV-1 subtype C for CCR5. To test this, we have characterized the interaction of gp120 envelope proteins from HIV-1 subtype C clones with CD4 and CCR5. Recombinant gp120 proteins from isolates of HIV-1 subtypes B and C were expressed, purified, and assessed in a CD4 binding assay and a CCR5 chemokine competition binding assay. All gp120 proteins bound to CD4-expressing cells, except one, 97ZA347ts, which had Arg substituted for the Cys239 in the conserved C2 loop. Reconstitution of Cys239, using site-directed mutagenesis, restored CD4 binding, while introducing Arg or Ser into position 239 of the functional Du151 gp120 protein abrogated CD4 binding. This shows that the Cys228-Cys239 disulfide bond of gp120 is required for high-affinity binding to CD4. Recombinant gp120 proteins from two HIV-1 subtype B clones bound CCR5 in the presence of CD4, while gp120 from the X4-tropic, HxB2, clone did not bind CCR5. gp120 from two functional HIV-1 subtype C clones, Du151 and MOLE1, bound CCR5 with high affinity in the presence of CD4 and Du151 showed significant CCR5 binding in the absence of CD4. A gp120 from a nonfunctional subtype C clone had lower affinity for CCR5. These results indicate that HIV-1 subtype C proteins have high affinity for CCR5 with variable dependence on CD4.

  1. Reconstitution of high-affinity binding of a beta-scorpion toxin to neurotoxin receptor site 4 on purified sodium channels.

    Science.gov (United States)

    Thomsen, W; Martin-Eauclaire, M F; Rochat, H; Catterall, W A

    1995-09-01

    Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1-3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a beta-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). 125I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min-1 and 0.015 min-1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with KD of approximately 0.1 nM and a major class with a KD of approximately 5 nM. Approximately 0.8 mol 125I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1-3 or 5, consistent with the characteristics of binding of beta-scorpion toxins to sodium channels in cells and membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase.

    Science.gov (United States)

    Hwang, Y; Feiss, M

    1996-08-30

    Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimulated endonuclease activity of terminase is located in gpA, the large terminase subunit. There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490. Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase. In the present work, mutations causing the gpA changes K497A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo. Purified K497A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in cos cleavage are sufficient to explain the lethality of the K497A and K497D defects. In in vitro packaging studies using mature (cleaved) phage DNA, the K497A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 microM ATP, indicating that the K497D change does not affect DNA translocation. In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase.

  3. Programmed Death-1 Culls Peripheral Accumulation of High-Affinity Autoreactive CD4 T Cells to Protect against Autoimmunity

    OpenAIRE

    2016-01-01

    Self-reactive CD4 T cells are incompletely deleted during thymic development, and their peripheral seeding highlights the need for additional safeguards to avert autoimmunity. Here, we show an essential role for the coinhibitory molecule programmed death-1 (PD-1) in silencing the activation of high-affinity autoreactive CD4 T cells. Each wave of self-reactive CD4 T cells that escapes thymic deletion autonomously upregulates PD-1 to maintain self-tolerance. By tracking the progeny derived from...

  4. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    Science.gov (United States)

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

  5. A new class of fluorescent boronic acids that have extraordinarily high affinities for diols in aqueous solution at physiological pH.

    Science.gov (United States)

    Cheng, Yunfeng; Ni, Nanting; Yang, Wenqian; Wang, Binghe

    2010-12-03

    The boronic acid group is an important recognition moiety for sensor design. Herein, we report a series of isoquinolinylboronic acids that have extraordinarily high affinities for diol-containing compounds at physiological pH. In addition, 5- and 8-isoquinolinylboronic acids also showed fairly high binding affinities towards D-glucose (K(a)=42 and 46 M(-1), respectively). For the first time, weak but encouraging binding of cis-cyclohexanediol was found for these boronic acids. Such binding was coupled with significant fluorescence changes. Furthermore, 4- and 6-isoquinolinylboronic acids also showed the ability to complex methyl α-D-glucopyranose (K(a)=3 and 2 M(-1), respectively).

  6. Synthesis, Characterization and Biological Activities of a New Fluorescent Indicator for the Intracellular Calcium Ions

    Institute of Scientific and Technical Information of China (English)

    HE Huaizhen; LEI Lei; LI Jianli; SHI Zhen

    2009-01-01

    A novel calcium-selective fluorescent indicator Fluo-3M AM was synthesized by introduction of a methyl group into the Ca2+-chelating moiety and adequately characterized by spectral methods (1H NMR, GC-MS, IR and MALDI-TOF MS). Meanwhile, its fluorescence spectra and some biological activities have been also studied. The results indicate that the new fluorescent indicator has relatively high affinity to calcium and a strong fluorescence signal, which should be useful for biomedical researchers to investigate the effects of calcium ions in biosystems.

  7. Effects of low phosphorus stress on iron,manganese,calcium and magnesium uptake in watermelon seedlings%低磷胁迫对西瓜苗期铁、锰、钙、镁吸收的影响

    Institute of Scientific and Technical Information of China (English)

    孟祥祥; 刘娜; 张莉; 杨景华; 张明方

    2011-01-01

    采用植物生长箱溶液培养方式,研究了低磷(P)胁迫对7个西瓜品种苗期铁(Fe)、锰(Mn)、钙(Ca)、镁(Mg)吸收的影响。结果表明,低磷胁迫会影响西瓜Fe,Mn,Ca,Mg的吸收。低磷胁迫使西瓜Fe的吸收降低,而对Mn、Ca和Mg吸收的影响则因品种而异。在低P胁迫条件下Ca,Mg的吸收表现出了极显著的正相关性。以相对元素含量为筛选指标研究低磷胁迫下7个品种Fe、Mn、Ca、Mg吸收的差异。结果表明,在低磷胁迫下,早佳和早春红玉为Fe高效吸收基因型,早佳为Mn高效吸收基因型,丽芳为Ca和Mg高效吸收基因型%Effects of low phosphorus(P) stress on iron(Fe),manganese(Mn),calcium(Ca) and magnesium(Mg) uptake in watermelon seedlings of seven cultivars were studied in this paper by the method of plant solution culture.It has been demonstrated that low P stress can affect the Fe,Mn,Ca and Mg uptake in the seven watermelon cultivars.Low P stress can reduce Fe uptake of watermelon,but the effects of low P stress on Mn,Ca and Mg uptake were different among cultivars.There were significant and positive correlation between Ca and Mg uptake under low P stress.The differences of Fe,Mn,Ca and Mg content among the seven cultivars were studied with the relative content of elements as screening parameters.Our results showed that Zaojia and Zaochunhongyu were identified as the Fe high efficient uptake genotypes,Zaojia was identified as the Mn high efficient uptake genotype,Lifang was identified as the Ca and Mg high efficient uptake genotype under low P stress.

  8. Programmed Death-1 Culls Peripheral Accumulation of High-Affinity Autoreactive CD4 T Cells to Protect against Autoimmunity

    Directory of Open Access Journals (Sweden)

    Tony T. Jiang

    2016-11-01

    Full Text Available Self-reactive CD4 T cells are incompletely deleted during thymic development, and their peripheral seeding highlights the need for additional safeguards to avert autoimmunity. Here, we show an essential role for the coinhibitory molecule programmed death-1 (PD-1 in silencing the activation of high-affinity autoreactive CD4 T cells. Each wave of self-reactive CD4 T cells that escapes thymic deletion autonomously upregulates PD-1 to maintain self-tolerance. By tracking the progeny derived from individual autoreactive CD4 T cell clones, we demonstrate that self-reactive cells with the greatest autoimmune threat and highest self-antigen affinity express the most PD-1. Reciprocally, PD-1 deprivation unleashes high-affinity self-reactive CD4 T cells in target tissues to exacerbate neuronal inflammation and autoimmune diabetes. Reliance on PD-1 to actively maintain self-tolerance may explain why exploiting this pathway by cancerous cells and invasive microbes efficiently subverts protective immunity, and why autoimmune side effects can develop after PD-1-neutralizing checkpoint therapies.

  9. Involvement of nitrogen functional groups in high-affinity copper binding in tomato and wheat root apoplasts: spectroscopic and thermodynamic evidence.

    Science.gov (United States)

    Guigues, Stéphanie; Bravin, Matthieu N; Garnier, Cédric; Masion, Armand; Chevassus-Rosset, Claire; Cazevieille, Patrick; Doelsch, Emmanuel

    2016-03-01

    Carboxylic groups located in plant cell walls (CW) are generally considered to be the main copper binding sites in plant roots, despite the presence of other functional groups. The aim of this study was to investigate sites responsible for copper binding in root apoplasts, i.e. CW and outer surface of the plasma membrane (PM) continuum. Binding sites in root apoplasts were investigated by comparing isolated CW of a monocotyledon (Triticum aestivum L.) and dicotyledon (Solanum lycopersicum L.) crop with their respective whole roots. Copper speciation was examined by X-ray absorption (XAS) and (13)C-nuclear magnetic resonance spectroscopies while the affinity of ligands involved in copper binding was investigated by modeling copper sorption isotherms. Homogeneous speciation and binding of copper was found in wheat and tomato root apoplasts. Only Cu-N and Cu-O bonds were detected in wheat and tomato root apoplasts. Nitrogen/oxygen ligands were identified in slightly higher proportions (40-70%) than single oxygen ligands. Furthermore, low- and high-affinity binding sites contributed in an almost equivalent proportion to copper binding in root apoplasts. The high-affinity N functional groups embedded in root apoplasts participated in copper binding in the same magnitude than the low-affinity carboxylic groups.

  10. Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: selective loss of agonist low-affinity and pirenzepine high-affinity binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Cioffi, C.L.; el-Fakahany, E.E.

    1986-09-01

    The effects of brief incubation with carbamylcholine on subsequent binding of (/sup 3/H)N-methylscopolamine were investigated in mouse neuroblastoma cells (clone N1E-115). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both pirenzepine and carbamylcholine interacted with high- and low-affinity subsets of muscarinic receptors. Computer-assisted analysis of the competition between pirenzepine and carbamylcholine with (/sup 3/H)N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various muscarinic receptor agonists to induce a decrease in consequent (/sup 3/H)N-methylscopolamine binding and their efficacy in stimulating cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in cyclic GMP levels were capable of diminishing ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of muscarinic receptor-mediated cyclic GMP formation.

  11. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Directory of Open Access Journals (Sweden)

    Bryan L Roth

    Full Text Available 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-methoxyphenylcyclohexanone and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenylcyclohexanamine and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenylcyclohexyl]piperidine and 1-[1-(4-methoxyphenylcyclohexyl]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.

  12. Effect of P Availability on Temporal Dynamics of Carbon Allocation and Glomus intraradices High-Affinity P Transporter Gene Induction in Arbuscular Mycorrhiza

    Science.gov (United States)

    Olsson, Pål Axel; Hansson, Maria C.; Burleigh, Stephen H.

    2006-01-01

    Arbuscular mycorrhizal (AM) fungi depend on a C supply from the plant host and simultaneously provide phosphorus to the colonized plant. We therefore evaluated the influence of external P on C allocation in monoxenic Daucus carota-Glomus intraradices cultures in an AM symbiosis. Fungal hyphae proliferated from a solid minimal medium containing colonized roots into a C-free liquid minimal medium with high or low P availability. Roots and hyphae were harvested periodically, and the flow of C from roots to fungus was measured by isotope labeling. We also measured induction of a G. intraradices high-affinity P transporter to estimate fungal P demand. The prevailing hypothesis is that high P availability reduces mycorrhizal fungal growth, but we found that C flow to the fungus was initially highest at the high P level. Only at later harvests, after 100 days of in vitro culture, were C flow and fungal growth limited at high P availability. Thus, AM fungi can benefit initially from P-enriched environments in terms of plant C allocation. As expected, the P transporter induction was significantly greater at low P availability and greatest in very young mycelia. We found no direct link between C flow to the fungus and the P transporter transcription level, which indicates that a good C supply is not essential for induction of the high-affinity P transporter. We describe a mechanism by which P regulates symbiotic C allocation, and we discuss how this mechanism may have evolved in a competitive environment. PMID:16751522

  13. Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    ¿-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain...... homogenate and slices. Our data show that [(125)I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([(125)I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (K(d), 7 nM; B(max), 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs......, but not by ¿-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites...

  14. Novel Radioiodinated γ-Hydroxybutyric Acid Analogues for Radiolabeling and Photolinking of High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a 125I-labeled GHB analog and characterized its binding in rat brain...... homogenate and slices. Our data show that [125I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([125I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (Kd, 7 nM; Bmax, 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ......-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites or [3H...

  15. Novel high-affinity and selective biaromatic 4-substituted gamma-hydroxybutyric acid (GHB) analogues as GHB ligands: design, synthesis, and binding studies.

    Science.gov (United States)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte; Frydenvang, Karla; Dahl, Ivar F; Bräuner-Osborne, Hans; Brehm, Lotte; Frølund, Bente; Clausen, Rasmus P

    2008-12-25

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites in brain, of which the latter have not been linked unequivocally to function, but are speculated to be GHB receptors. In this study, a series of biaromatic 4-substituted GHB analogues, including 4'-phenethylphenyl, 4'-styrylphenyl, and 4'-benzyloxyphenyl GHB analogues, were synthesized and characterized pharmacologically in a [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([3H]NCS-382) binding assay and in GABA(A) and GABA(B) receptor binding assays. The compounds were selective for the high-affinity GHB binding sites and several displayed Ki values below 100 nM. The affinity of the 4-[4'-(2-iodobenzyloxy)phenyl] GHB analogue 17b was shown to reside predominantly with the R-enantiomer (Ki = 22 nM), which has higher affinity than previously reported GHB ligands.

  16. Michael Acceptor Approach to the Design of New Salvinorin A-based High Affinity Ligands for the Kappa-Opioid Receptor

    Science.gov (United States)

    Polepally, Prabhakar R.; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D.; Roth, Bryan L.; Zjawiony, Jordan K.

    2014-01-01

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand–receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure–activity relationships, and previous salvinorin A–KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR. PMID:25193297

  17. Michael acceptor approach to the design of new salvinorin A-based high affinity ligands for the kappa-opioid receptor.

    Science.gov (United States)

    Polepally, Prabhakar R; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D; Roth, Bryan L; Zjawiony, Jordan K

    2014-10-06

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand-receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure-activity relationships, and previous salvinorin A-KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR.

  18. Influence of ischemic preconditioning on levels of nerve growth factor, brain-derived neurotrophic factor and their high-affinity receptors in hippocampus following forebrain ischemia.

    Science.gov (United States)

    Lee, Tsong-Hai; Yang, Jen-Tsung; Ko, Yu-Shien; Kato, Hiroyuki; Itoyama, Yasuto; Kogure, Kyuya

    2008-01-02

    Preconditioning of gerbil brain with a sublethal forebrain ischemia is known to protect hippocampal CA1 neurons following a subsequent lethal ischemia (the second ischemia) which usually damages neurons (ischemic tolerance). Present report using a confocal laser scanning microscope demonstrated that the hippocampal cells of sham operation gerbils contained immunofluorescent NGF and BDNF and their high-affinity receptors (TrkA and TrkB). A 2-min ischemia caused little change of these proteins (ANOVA test, PBDNF but not NGF and their high-affinity receptors showed a transient reduction at 4 h (ANOVA test, PBDNF and TrkB decreased transiently from 4 h to 1 day (ANOVA test, PCA3 and dentate gyrus areas, only BDNF decreased significantly at 7 days in the CA3 area without ischemic preconditioning (ANOVA test, PCA3 and dentate gyrus areas with and without ischemic preconditioning. Western blot study showed that in the hippocampal formation with ischemic preconditioning, preconditioning prevented the decline of these protein levels from 1 day to 7 days after the second lethal ischemia (ANOVA test, P>0.05). Results of this study demonstrate that ischemic preconditioning recovers the initial decline in NGF and BDNF and their corresponding receptors in the vulnerable CA1 neurons after the second lethal ischemia, suggesting that growth factors might play a role in the protective mechanism of ischemic preconditioning.

  19. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H. (J. Plateaustraat, Gent (Belgium))

    1988-11-01

    Binding studies were performed with two {sup 125}I-labeled Bacillus thuringiensis {delta}-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One {delta}-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other {delta}-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis {delta}-endotoxins active against M. sexta compete for binding of {sup 125}I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.

  20. The high-affinity maltose switch MBP317-347 has low affinity for glucose: implications for targeting tumors with metabolically directed enzyme prodrug therapy.

    Science.gov (United States)

    Valdes, Gilmer; Schulte, Reinhard W; Ostermeier, Marc; Iwamoto, Keisuke S

    2014-03-01

    Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

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

    Science.gov (United States)

    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.

  2. 2-Nitroimipramine: a photoaffinity probe for the serotonin uptake/tricyclic binding site complex.

    OpenAIRE

    Wennogle, L P; Ashton, R A; Schuster, D. I.; Murphy, R B; Meyerson, L R

    1985-01-01

    [3H]2-Nitroimipramine ([3H]2-NI), a compound with high affinity for the serotonin uptake system, is shown to be an effective photoaffinity probe which incorporates covalently into membrane homogenates prepared from human platelets, as well as rat brain and liver. In all cases, [3H]2-NI preferentially incorporated into a minor membrane component of 30 kd protein, as determined by SDS-polyacrylamide gel electrophoresis and subsequent fluorography. A number of selective and general serotonin upt...

  3. Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Sabbatini, Paola; Wellendorph, Petrine; Høg, Signe;

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report...... the design and synthesis of the first 125I-labeled radioligands in the field, one of which contains a photoaffinity label which enables it to bind irreversibly to the high-affinity GHB binding sites....

  4. Calcium in diet

    Science.gov (United States)

    ... D is needed to help your body use calcium. Milk is fortified with vitamin D for this reason. ... of calcium dietary supplements include calcium citrate and calcium carbonate. Calcium citrate is the more expensive form of ...

  5. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  6. Comparison of high affinity binding of {sup 3}H-proadifen and {sup 3}H-(-)-cocaine t rat liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B. [Astra Arcus AB, Dept. of Neuropharmacology, Soedertaelje (Sweden)

    1995-06-01

    The characteristics of the binding of {sup 3}H-proadifen to rat liver membranes were studied and compared to those of {sup 3}H-cocaine. It was found that {sup 3}H-proadifen was bound reversibly with high affinity (K{sub D}=1.8{+-}0.5 nM) and large capacity (B{sub max}=2010{+-}340 pmol/g wet tissue) to liver membranes. The corresponding values for the {sup 3}H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of {sup 3}H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 {mu}M CdCl{sub 2} in the incubation buffer it was possible to differentiate between two {sup 3}H-cocaine binding sites with K{sub d} values of 1.6 and 7.7 nM and B{sub max} values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of {sup 3}H-proadifen and {sup 3}H-cocaine inhibited the binding of {sup 3}H-proadifen (IC{sub 50}=10 nM) and proadifen that of {sup 3}H-cocaine (IC{sub 50}=1 nM). There was a high correlation coefficient (r{sub r}=0.972; P<0.01; n=12) in the Spearman rank test between the inhibitory potencies of compounds examined in both systems. Beside some potent alaproclate analogues a couple of compounds had moderately high affinity (IC{sub 50}=100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl{sub 2}, ZnCl{sub 2} and CuCl{sub 2} inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd{sup 2+} on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15{mu}M. The similarity of the characteristics of the binding of these ligands with that of {sup 3}H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.).

  7. Calcium supplements

    Science.gov (United States)

    ... Related Bone Diseases National Resource Center. Calcium and vitamin D: Important at every age. NIAMS.NIH.gov website. www.niams.nih.gov/Health_Info/Bone/Bone_Health/Nutrition . Updated May 2015. Accessed March ...

  8. Calcium Electroporation

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha;

    2015-01-01

    BACKGROUND: Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill...... efficacy-and normal cell sensitivity. METHODS: Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780......), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n). RESULTS: The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p

  9. Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Z.; Chen, S.; Wilson, D.B.

    1999-11-01

    An Mn{sup 2+} and Cd{sup 2+} uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd{sup 2+} sensitivity as well as energy-dependent Cd{sup 2+} uptake activity. Both transcription and translation of mntA were induced by Mn{sup 2+} starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd{sup 2+} uptake systems have been identified in L. plantarum: one is a high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system that is expressed in Mn{sup 2+}-starved cells, and the other is a nonsaturable Cd{sup 2+} uptake system that is expressed in Cd{sup 2+}-sufficient cells. MntA was not detected in an Mn{sup 2+}-dependent mutant of L. plantarum which had lost high-affinity Mn{sup 2+} and Cd{sup 2+} uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn{sup 2+} and Cd{sup 2+} transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn{sup 2+}- and Cd{sup 2+}-binding sites of MntA are discussed. A second clone containing a low-affinity Cd{sup 2+} transport system was also isolated.

  10. LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

    Science.gov (United States)

    Fliegmann, Judith; Jauneau, Alain; Pichereaux, Carole; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Burlet-Schiltz, Odile; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-01

    LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.

  11. A novel rabbit immunospot array assay on a chip allows for the rapid generation of rabbit monoclonal antibodies with high affinity.

    Directory of Open Access Journals (Sweden)

    Tatsuhiko Ozawa

    Full Text Available Antigen-specific rabbit monoclonal antibodies (RaMoAbs are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10(-12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.

  12. Design, Synthesis, Binding and Docking-Based 3D-QSAR Studies of 2-Pyridylbenzimidazoles—A New Family of High Affinity CB1 Cannabinoid Ligands

    Directory of Open Access Journals (Sweden)

    Patricio Iturriaga-Vásquez

    2013-04-01

    Full Text Available A series of novel 2-pyridylbenzimidazole derivatives was rationally designed and synthesized based on our previous studies on benzimidazole 14, a CB1 agonist used as a template for optimization. In the present series, 21 compounds displayed high affinities with Ki values in the nanomolar range. JM-39 (compound 39 was the most active of the series (KiCB1 = 0.53 nM, while compounds 31 and 44 exhibited similar affinities to WIN 55212-2. CoMFA analysis was performed based on the biological data obtained and resulted in a statistically significant CoMFA model with high predictive value (q2 = 0.710, r2 = 0.998, r2pred = 0.823.

  13. The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein.

    Science.gov (United States)

    Kozyraki, R; Fyfe, J; Kristiansen, M; Gerdes, C; Jacobsen, C; Cui, S; Christensen, E I; Aminoff, M; de la Chapelle, A; Krahe, R; Verroust, P J; Moestrup, S K

    1999-06-01

    Cubilin is the intestinal receptor for the endocytosis of intrinsic factor-vitamin B12. However, several lines of evidence, including a high expression in kidney and yolk sac, indicate it may have additional functions. We isolated apolipoprotein A-I (apoA-I), the main protein of high-density lipoprotein (HDL), using cubilin affinity chromatography. Surface plasmon resonance analysis demonstrated a high-affinity binding of apoA-I and HDL to cubilin, and cubilin-expressing yolk sac cells showed efficient 125I-HDL endocytosis that could be inhibited by IgG antibodies against apoA-I and cubilin. The physiological relevance of the cubilin-apoA-I interaction was further emphasized by urinary apoA-I loss in some known cases of functional cubilin deficiency. Therefore, cubilin is a receptor in epithelial apoA-I/HDL metabolism.

  14. Impaired signaling via the high-affinity IgE receptor in Wiskott-Aldrich syndrome protein-deficient mast cells.

    Science.gov (United States)

    Pivniouk, Vadim I; Snapper, Scott B; Kettner, Alexander; Alenius, Harri; Laouini, Dhafer; Falet, Hervé; Hartwig, John; Alt, Frederick W; Geha, Raif S

    2003-12-01

    Wiskott-Aldrich syndrome protein (WASP) is the product of the gene deficient in boys with X-linked Wiskott-Aldrich syndrome. We assessed the role of WASP in signaling through the high-affinity IgE receptor (FcepsilonRI) using WASP-deficient mice. IgE-dependent degranulation and cytokine secretion were markedly diminished in bone marrow-derived mast cells from WASP-deficient mice. Upstream signaling events that include FcepsilonRI-triggered total protein tyrosine phosphorylation, and protein tyrosine phosphorylation of FcepsilonRIbeta and Syk were not affected by WASP deficiency. However, tyrosine phosphorylation of phospholipase Cgamma and Ca(2+) mobilization were diminished. IgE-dependent activation of c-Jun N-terminal kinase, cell spreading and redistribution of cellular F-actin in mast cells were reduced in the absence of WASP. We conclude that WASP regulates FcepsilonRI-mediated granule exocytosis, cytokine production and cytoskeletal changes in mast cells.

  15. Enzyme-amplified protein micorarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Varnum, Susan M.; Warner, Marvin G.; Dockendorff, Brian P.; Anheier, Norman C.; Lou, Jianlong; Marks, James D.; Smith, Leonard A.; Feldhaus, Michael J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2006-06-16

    With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay, because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.

  16. Drug binding to the inactivated state is necessary but not sufficient for high-affinity binding to human ether-à-go-go-related gene channels.

    Science.gov (United States)

    Perrin, Mark J; Kuchel, Philip W; Campbell, Terence J; Vandenberg, Jamie I

    2008-11-01

    Drug block of the human ether-à-go-go-related gene K(+) channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.

  17. Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain.

    Science.gov (United States)

    Vu, Trang T; Stafford, Alan R; Leslie, Beverly A; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I

    2011-09-01

    Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.

  18. Crystallization and preliminary X-ray structural studies of a high-affinity CD8αα co-receptor to pMHC

    Energy Technology Data Exchange (ETDEWEB)

    Cole, David K. [Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU (United Kingdom); Rizkallah, Pierre J., E-mail: p.j.rizkallah@dl.ac.uk [CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD (United Kingdom); Sami, Malkit; Lissin, Nikolai M.; Gao, Feng [Avidex Ltd, 57c Milton Park, Abingdon, Oxon OX14 4RX (United Kingdom); Bell, John I. [Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU (United Kingdom); Boulter, Jonathan M. [Medical Biochemistry and Immunology, Henry Wellcome Building, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN,Wales (United Kingdom); Glick, Meir [Novartis Pharmaceuticals, One Health Plaza, East Hanover, NJ 07936 (United States); Vuidepot, Anne-Lise; Jakobsen, Bent K., E-mail: p.j.rizkallah@dl.ac.uk [Avidex Ltd, 57c Milton Park, Abingdon, Oxon OX14 4RX (United Kingdom); Gao, George F. [Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU (United Kingdom)

    2005-03-01

    A high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. The class I CD8 positive T-cell response is involved in a number of conditions in which artificial down-regulation and control would be therapeutically beneficial. Such conditions include a number of autoimmune diseases and graft rejection in transplant patients. Although the CD8 T-cell response is dominated by the TCR–pMHC interaction, activation of T cells is in most cases also dependent on a number of associated signalling molecules. Previous work has demonstrated the ability of one such molecule (CD8) to act as an antagonist to T-cell activation if added in soluble form. Therefore, a high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. Single haCD8 crystals were cryocooled and used for data collection. These crystals belonged to space group P6{sub 4}22 (assumed by similarity to the wild type), with unit-cell parameters a = 101.08, c = 56.54 Å. V{sub M} calculations indicated one molecule per asymmetric unit. A 2 Å data set was collected and the structure is currently being determined using molecular replacement.

  19. Structure-affinity properties of a high-affinity ligand of FKBP12 studied by molecular simulations of a binding intermediate.

    Directory of Open Access Journals (Sweden)

    Lilian Olivieri

    Full Text Available With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant [Formula: see text] of this intermediate should be decreased, resulting in an increase of the affinity constant [Formula: see text]. The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state.

  20. The Interaction of Arp2/3 Complex with Actin: Nucleation, High Affinity Pointed End Capping, and Formation of Branching Networks of Filaments

    Science.gov (United States)

    Dyche Mullins, R.; Heuser, John A.; Pollard, Thomas D.

    1998-05-01

    The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μ M. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

  1. High affinity binding of /sup 125/I-labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties

    Energy Technology Data Exchange (ETDEWEB)

    Aguet, M.; Blanchard, B.

    1981-12-01

    Direct ligand-binding studies with highly purified /sup 125/I-labeled virus-induced mouse interferon on mouse lymphoma L 1210 cells revealed a direct correlation of specific high-affinity binding with the biologic response to interferon. Neutralization of the antiviral effect by anti-interferon gamma globulin occurred at the same antibody concentration as the inhibition of specific binding. These results suggest that specific high-affinity binding of /sup 125/I-interferon occurred at a biologically functional interferon receptor. Competitive inhibition experiments using /sup 125/I- and /sup 127/I-labeled interferon provided strong evidence that the fraction of /sup 125/I-interferon inactivated upon labeling did not bind specifically. Scatchard analysis of the binding data yielded linear plots and thus suggested that interferon binds to homogeneous noncooperative receptor sites. In contrast to a characteristic property of several peptide hormone systems, binding of /sup 125/I-interferon to its specific receptor did not induce subsequent ligand degradation. At 37/sup o/ bound interferon was rapidly released in a biologically active form without evidence for molecular degradation. The expression of interferon receptors was not modified by treatment with interferon. Trypsin treatment of target cells and inhibition of protein synthesis abolished the specific binding of /sup 125/I-interferon. Three major molecular weight species of Newcastle disease virus-induced mouse C 243 cell interferon were isolated, separated, and identified as mouse ..cap alpha.. and ..beta.. interferons. These interferons were shown to inhibit competitively the specific binding of the highly purified labeled starting material thus providing evidence for a common receptor site for mouse interferon.

  2. Discovery of high affinity ligands for β2-adrenergic receptor through pharmacophore-based high-throughput virtual screening and docking.

    Science.gov (United States)

    Yakar, Ruya; Akten, Ebru Demet

    2014-09-01

    Novel high affinity compounds for human β2-adrenergic receptor (β2-AR) were searched among the clean drug-like subset of ZINC database consisting of 9,928,465 molecules that satisfy the Lipinski's rule of five. The screening protocol consisted of a high-throughput pharmacophore screening followed by an extensive amount of docking and rescoring. The pharmacophore model was composed of key features shared by all five inactive states of β2-AR in complex with inverse agonists and antagonists. To test the discriminatory power of the pharmacophore model, a small-scale screening was initially performed on a database consisting of 117 compounds of which 53 antagonists were taken as active inhibitors and 64 agonists as inactive inhibitors. Accordingly, 7.3% of the ZINC database subset (729,413 compounds) satisfied the pharmacophore requirements, along with 44 antagonists and 17 agonists. Afterwards, all these hit compounds were docked to the inactive apo form of the receptor using various docking and scoring protocols. Following each docking experiment, the best pose was further evaluated based on the existence of key residues for antagonist binding in its vicinity. After final evaluations based on the human intestinal absorption (HIA) and the blood brain barrier (BBB) penetration properties, 62 hit compounds have been clustered based on their structural similarity and as a result four scaffolds were revealed. Two of these scaffolds were also observed in three high affinity compounds with experimentally known Ki values. Moreover, novel chemical compounds with distinct structures have been determined as potential β2-AR drug candidates.

  3. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1

    DEFF Research Database (Denmark)

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-01-01

    Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake...... of verapamil. Fluxes of radiolabelled verapamil across MDCK II MDR1 monolayers were measured in the absence and presence of the putative P-gp inhibitor telmisartan (a clinically approved drug compound). Verapamil displayed a vectorial basolateral-to-apical transepithelial efflux across the MDCK II MDR1...

  4. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1.

    Science.gov (United States)

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-08-01

    Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake of verapamil. Fluxes of radiolabelled verapamil across MDCK II MDR1 monolayers were measured in the absence and presence of the putative P-gp inhibitor telmisartan (a clinically approved drug compound). Verapamil displayed a vectorial basolateral-to-apical transepithelial efflux across the MDCK II MDR1 monolayers with a permeability of 5.7 × 10(-5) cm sec(-1) compared to an apical to basolateral permeability of 1.3 × 10(-5) cm sec(-1). The efflux could be inhibited with the P-gp inhibitor zosuquidar. Zosuquidar (0.4 μmol/L) reduced the efflux ratio (PB-A/PA-B) for verapamil 4.6-1.6. The presence of telmisartan, however, only caused a slight reduction in P-gp-mediated verapamil transport to an efflux ratio of 3.4. Overall, the results of the present in vitro approach indicate, that clinical use of telmisartan as a P-gp inhibitor may not be an effective strategy for increasing brain uptake of verapamil by co-administration with telmisartan.

  5. Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

    Science.gov (United States)

    Hou, Bi-Huei; Takanaga, Hitomi; Griesbeck, Oliver; Frommer, Wolf B

    2009-08-01

    Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. Here, the high sensitivity FRET calcium sensor TN-XXL was used to characterize glucose-induced calcium responses in non-excitable human embryonic kidney HEK293T cells. HEK293T cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels. Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEK293T cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced. Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity.

  6. Calcium Signaling Is Required for Erythroid Enucleation.

    Science.gov (United States)

    Wölwer, Christina B; Pase, Luke B; Russell, Sarah M; Humbert, Patrick O

    2016-01-01

    Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation.

  7. Calcium Signaling Is Required for Erythroid Enucleation.

    Directory of Open Access Journals (Sweden)

    Christina B Wölwer

    Full Text Available Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation.

  8. Effects of calcium on growth of winter wheat seedlings and nutrient uptake under partial-root water stress%局部根区水分胁迫下钙对冬小麦生长及养分吸收的影响

    Institute of Scientific and Technical Information of China (English)

    周芳; 赵玉霞; 王文岩; 李雪芳; 王林权

    2015-01-01

    set up in this experiment ,including a factorial combina-tion of three water stress schemes (no water stress (CK ) ,half-root water stress (HRWS ) and total-root water stress (TR-WS)) and two calcium treatments (with calcium and without ) .The results showed that shoot height ,taproot length , biomass ,relative water content (RWC ) ,relative chlorophyll content (SPAD ) ,and N ,P ,Ca concentrations in winter wheat seedlings without calcium treatment were significantly lower than those with calcium ,regardless of water stress ( P<0 .05 ) .With the treatment of calcium ,plant height in HRWS was increased by 4 .4% .Biomass in CK ,HRWS ,and TRWS were 1 .54 ,1 .66 g·plant-1 and 0 .97 g·plant-1 ,respectively ,which were 19 .4% ,25 .8% and 4 .3% greater than those without treatment of calcium .Plant height and root length in TRWS became decreased significantly .Without the treatment of calcium ,concentrations of N ,P and Ca etc .in seedlings were significantly reduced .Total nitrogen of shoot with calcium treatment in CK ,HRWS ,and TRWS were 36 .54 ,36 .65 g·kg-1 and 32 .70 g·kg-1 ,9 .5% ,6 .5%and 6 .9% ,respectively ,higher than those without;total phosphorus were 7 .48 ,7 .51 g·kg-1 and 6 .54 g·kg-1 , 3 .0% ,13 .1% and 22 .7% ,respectively ,higher than those without;above-ground total calcium were 8 .35 ,8 .37 g·kg-1 and 5 .53 g·kg-1 respectively ,26 .5% ,24 .4% and 19 .7% higher than those without calcium .The results in-dicated that calcium had significant effects on the development and nutrient use of winter wheat seedlings by promoting growth and nutrient uptake under water stress ,relieving the inhibitory effect in the entire root .

  9. Calcium electrotransfer for termination of transgene expression in muscle

    DEFF Research Database (Denmark)

    Hojman, Pernille; Spanggaard, Iben; Olsen, Caroline Holkman

    2011-01-01

    . A clinical grade calcium solution (20 μl, 168 mM) was injected into transfected mouse or rat tibialis cranialis muscle. Ca(2+) uptake was quantified using calcium 45 ((45)Ca), and voltage and time between injection and pulsation were varied. Extinction of transgene expression was investigated by using both...

  10. Calcium and bones

    Science.gov (United States)

    Bone strength and calcium ... calcium (as well as phosphorus) to make healthy bones. Bones are the main storage site of calcium in ... your body does not absorb enough calcium, your bones can get weak or will not grow properly. ...

  11. Uptake of anionic radionuclides onto degraded cement pastes and competing effect of organic ligands

    Energy Technology Data Exchange (ETDEWEB)

    Pointeau, I.; Coreau, N. [L3MR, Lab. de Mesures et Modelisation de la Migration des Radionucleides, CEA Saclay, Direction de l' Energie Nucleaire/Dept. de Physico-Chimie/Service d' Etude du Comportement des Radionucleides, Gif-sur-Yvette (France); Reiller, P.E. [LSRM, Lab. de Speciation des Radionucleides et des Molecules, CEA Saclay, Direction de l' Energie Nucleaire/Dept. de Physico-Chimie/Service d' Etude du Comportement des Radionucleides, Gif-sur Yvette (France)

    2008-07-01

    Hardened cement pastes (HCP) present a high affinity with a lot of radionuclides (RN) and can be used as waste confining materials in radioactive waste repository. Indeed, in cementitious media, RN can be removed from solution via (co)precipitation reactions or via sorption/diffusion mechanisms. In this study, the affinity of anionic RN (Cl{sup -}, I{sup -}, SeO{sub 3}{sup 2-} and CO{sub 3}{sup 2-} chemical forms) with a CEM-I HCP has been studied vs. the degradation of the HCP particles. These RN are considered as mobile in repository media and it is important to have a set of distribution ratio (R{sub d}) in cement environment. The R{sub d} values have been measured in batch experiments as a function of the pH, used as the degraded state parameter of the HCP suspensions. The R{sub d} values increase in all cases, from the unaltered state (pH 13.3) to the altered state of HCP, i.e. until all portlandite is dissolved, corresponding to pH 12.6. Then, R{sub d} values decrease until degraded states (pH 12.0), corresponding to the decalcification of the calcium silicate hydrate (C-S-H) phases. The behaviour of anionic RN seems to be correlated to the evolution of calcium concentration and is opposed to the evolution of sulphate concentration in solution which could have a competing effect. Comparison is done with the behaviour of caesium and uranium(VI), which is a cationic RN but has a major negative hydrolysed species at high pH. As awaited, the uranium(VI) behaviour is very different from purely anionic RN one in accord with spectroscopic analyses from literature works. The R{sub d} values have also been measured for the organic ligands isosaccharinate (ISA) and EDTA. The uptake of ISA can be important and competing effect with the sorption of SeO{sub 3}{sup 2-} has been evidenced in HCP suspensions as a function of the ISA concentration. (orig.)

  12. Calcium Carbonate

    Science.gov (United States)

    ... doctor if you have or have ever had kidney disease or stomach conditions.tell your doctor if you are pregnant, plan to become pregnant, or are breast-feeding. If you become pregnant while taking calcium carbonate, call your doctor.

  13. Calcium Test

    Science.gov (United States)

    ... if a person has symptoms of a parathyroid disorder , malabsorption , or an overactive thyroid. A total calcium level is often measured as part of a routine health screening. It is included in the comprehensive metabolic panel (CMP) and the basic metabolic panel (BMP) , ...

  14. An HIV-1 encoded peptide mimics the DNA binding loop of NF-{kappa}B and binds thioredoxin with high affinity

    Energy Technology Data Exchange (ETDEWEB)

    Su Guoping [Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352 (United States)]. E-mail: gsu@u.washington.edu; Wang Min [Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023 (United States)]. E-mail: wang.min@yale.edu; Taylor, Ethan Will [Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352 (United States)]. E-mail: wtaylor@rx.uga.edu

    2005-11-11

    Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-{kappa}B), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-{kappa}B via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-{kappa}B/Rel family of transcription factors by the putative HIV-1 pro-fs protein.

  15. High affinity of anti-GBM antibodies from Goodpasture and transplanted Alport patients to alpha3(IV)NC1 collagen.

    Science.gov (United States)

    Rutgers, A; Meyers, K E; Canziani, G; Kalluri, R; Lin, J; Madaio, M P

    2000-07-01

    Anti-glomerular basement membrane (anti-GBM) antibody-mediated diseases are characterized by rapidly progressive glomerulonephritis (RPGN) that often results in irreversible loss of renal function and renal failure. Although many factors contribute to the fulminant nature and treatment resistance of this disease, we questioned whether high affinity autoantibody-alpha3(IV) collagen interactions lead to persistent antibody deposition, thereby perpetuating inflammation. To address this hypothesis, the binding kinetics of human anti-GBM antibodies (Ab) to alpha3(IV)NC1 were evaluated using an optical biosensor interaction analysis. Polyclonal anti-GBM Abs were purified by alpha3(IV)NC1 affinity chromatography from the sera of patients with anti-GBM AB-mediated diseases, including individuals with Goodpasture syndrome (GS), idiopathic RPGN (N = 7), and Alport syndrome (AL) following kidney transplantation (N = 4). The affinity-binding characteristics of the autoantibodies were determined using a biosensor analysis system, with immobilized bovine alpha3(IV)NC1 dimers. All of the autoantibody preparations bound to alpha3(IV)NC1, whereas none bound to alpha1(IV)NC1 (control). Purified, normal serum IgG did not bind to either antigen. Estimated dissociation constants (Kd) for the purified autoantibodies were 1.39E-04 +/- 7.30E-05 s-l (GS) and 8. 90E-05 +/- 2.80E-05 s-l (AL). Their estimated association constants (Ka) were 2.67E+04 +/- 1.8E+04 (M-ls-l) and 2.76E+04 +/- 1. 70E+04(M-ls-l) for GS and AL patients, respectively. By comparison with other Ab interactions, these Abs demonstrated high affinity, with relatively high on (binding) rates and slow off (dissociation) rates. The results suggest that anti-GBM Abs bind rapidly and remain tightly bound to the GBM in vivo. This property likely contributes to both the fulminant nature of this disease and its resistance to therapy, because persistent glomerular Ab deposition has the potential to produce continuous inflammation

  16. Serum Proteins Stabilized Calcium Phosphate Nanoparticles and Its Effect on Bel-7402 Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Hydroxyapatite has a high affinity to biological macromolecules, especially to proteins. Bovine serum proteins were extracted to be used as stablizer to prepare calcium phosphate nanoparticles. 167.7 am and87.7 nm particles were respectively prepared by using bovine serum protein fractions at the concentration of 0.5mg/mL and 1.0 mg/mL. As the polysaccharide stabilized hydroxyapatite nanoparticles, the protein-stablized nanoparticles also inhibited the proliferation rate of Bel-7402 cells. It suggested that proteins could be applied to prepare calcium phosphate nanoparticles and it also has the anticancer effect.

  17. Intra-amniotic administration (Gallus gallus) of cicer arietinum and lens culinaris prebiotics extracts and duck egg white peptides affects calcium status and intestinal functionality

    Science.gov (United States)

    Calcium (Ca) is one of the most abundant inorganic elements in the human body and has many important physiological roles. Prebiotics and bioactive peptides are two important substances used to promote calcium uptake. However, the difference in mechanisms of the calcium uptake from these two suppleme...

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

    Science.gov (United States)

    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.

  19. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.

    Science.gov (United States)

    Xu, Qiang; Wang, Yanshu; Dabdoub, Alain; Smallwood, Philip M; Williams, John; Woods, Chad; Kelley, Matthew W; Jiang, Li; Tasman, William; Zhang, Kang; Nathans, Jeremy

    2004-03-19

    Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. We show here that Norrin and Fz4 function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors.

  20. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

    Energy Technology Data Exchange (ETDEWEB)

    Klein, M.; Canoll, P.D.; Musacchio, J.M. (New York Univ. Medical Center, New York, NY (USA))

    1991-01-01

    The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.

  1. The Mitochondrial Metallochaperone SCO1 Is Required to Sustain Expression of the High-Affinity Copper Transporter CTR1 and Preserve Copper Homeostasis

    Directory of Open Access Journals (Sweden)

    Christopher J. Hlynialuk

    2015-02-01

    Full Text Available Human SCO1 fulfills essential roles in cytochrome c oxidase (COX assembly and the regulation of copper (Cu homeostasis, yet it remains unclear why pathogenic mutations in this gene cause such clinically heterogeneous forms of disease. Here, we establish a Sco1 mouse model of human disease and show that ablation of Sco1 expression in the liver is lethal owing to severe COX and Cu deficiencies. We further demonstrate that the Cu deficiency is explained by a functional connection between SCO1 and CTR1, the high-affinity transporter that imports Cu into the cell. CTR1 is rapidly degraded in the absence of SCO1 protein, and we show that its levels are restored in Sco1−/− mouse embryonic fibroblasts upon inhibition of the proteasome. These data suggest that mitochondrial signaling through SCO1 provides a post-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they further underpin the importance of mitochondria in cellular Cu homeostasis.

  2. H2-saturation of high affinity H2-oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups.

    Science.gov (United States)

    Piché-Choquette, Sarah; Tremblay, Julien; Tringe, Susannah G; Constant, Philippe

    2016-01-01

    Soil microbial communities are continuously exposed to H2 diffusing into the soil from the atmosphere. N2-fixing nodules represent a peculiar microniche in soil where H2 can reach concentrations up to 20,000 fold higher than in the global atmosphere (0.530 ppmv). In this study, we investigated the impact of H2 exposure on soil bacterial community structure using dynamic microcosm chambers simulating soil H2 exposure from the atmosphere and N2-fixing nodules. Biphasic kinetic parameters governing H2 oxidation activity in soil changed drastically upon elevated H2 exposure, corresponding to a slight but significant decay of high affinity H2-oxidizing bacteria population, accompanied by an enrichment or activation of microorganisms displaying low-affinity for H2. In contrast to previous studies that unveiled limited response by a few species, the relative abundance of 958 bacterial ribotypes distributed among various taxonomic groups, rather than a few distinct taxa, was influenced by H2 exposure. Furthermore, correlation networks showed important alterations of ribotype covariation in response to H2 exposure, suggesting that H2 affects microbe-microbe interactions in soil. Taken together, our results demonstrate that H2-rich environments exert a direct influence on soil H2-oxidizing bacteria in addition to indirect effects on other members of the bacterial communities.

  3. Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria

    Science.gov (United States)

    Porcherie, Adeline; Mathieu, Cedric; Peronet, Roger; Schneider, Elke; Claver, Julien; Commere, Pierre-Henri; Kiefer-Biasizzo, Hélène; Karasuyama, Hajime; Milon, Geneviève; Dy, Michel; Kinet, Jean-Pierre; Louis, Jacques; Blank, Ulrich

    2011-01-01

    The role of the IgE–FcεRI complex in malaria severity in Plasmodium falciparum–hosting patients is unknown. We demonstrate that mice genetically deficient for the high-affinity receptor for IgE (FcεRIα-KO) or for IgE (IgE-KO) are less susceptible to experimental cerebral malaria (ECM) after infection with Plasmodium berghei (PbANKA). Mast cells and basophils, which are the classical IgE-expressing effector cells, are not involved in disease as mast cell–deficient and basophil-depleted mice developed a disease similar to wild-type mice. However, we show the emergence of an FcεRI+ neutrophil population, which is not observed in mice hosting a non–ECM-inducing PbNK65 parasite strain. Depletion of this FcεRI+ neutrophil population prevents ECM, whereas transfer of this population into FcεRIα-KO mice restores ECM susceptibility. FcεRI+ neutrophils preferentially home to the brain and induce elevated levels of proinflammatory cytokines. These data define a new pathogenic mechanism of ECM and implicate an FcεRI-expressing neutrophil subpopulation in malaria disease severity. PMID:21967768

  4. Site-specific conjugation of an antibody-binding protein catalyzed by horseradish peroxidase creates a multivalent protein conjugate with high affinity to IgG.

    Science.gov (United States)

    Minamihata, Kosuke; Goto, Masahiro; Kamiya, Noriho

    2015-01-01

    Cross-linking proteins offers an approach to enhance the distinct function of proteins due to the multivalent effect. In this study, we demonstrated the preparation of a multivalent antibody-binding protein possessing high affinity to IgG by conjugating a number of antibody-binding proteins using the horseradish peroxidase (HRP)-mediated protein conjugation method. By introducing a peptide tag containing a tyrosine (Y-tag) to the C-terminus of the model protein, a chimera protein of protein G and protein A (pG2 pA), the Tyr residue in the Y-tag was efficiently recognized by HRP and cross-linked with each other to yield a pG2 pA conjugate, composed of mainly two to three units of pG2 pA. The cross-linking occurred site specifically at the Tyr residue in the Y-tag and introduction of the Y-tag showed no effect on the function of pG2 pA. The affinity of the Y-tagged pG2 pA conjugate against IgG clearly increased because of the multivalent effect, demonstrating the benefit of this protein cross-linking reaction, which yields functional protein oligomers. Such multivalent protein conjugates created by this reaction should have potential to be used in ELISA and Western blotting applications in which highly sensitive detection of target molecules is desired.

  5. High-affinity Near-infrared Fluorescent Small-molecule Contrast Agents for In Vivo Imaging of Prostate-specific Membrane Antigen

    Directory of Open Access Journals (Sweden)

    Valerie Humblet

    2005-10-01

    Full Text Available Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM, single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

  6. High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice.

    Science.gov (United States)

    Boateng, Comfort A; Bakare, Oluyomi M; Zhan, Jia; Banala, Ashwini K; Burzynski, Caitlin; Pommier, Elie; Keck, Thomas M; Donthamsetti, Prashant; Javitch, Jonathan A; Rais, Rana; Slusher, Barbara S; Xi, Zheng-Xiong; Newman, Amy Hauck

    2015-08-13

    The dopamine D3 receptor (D3R) is a promising target for the development of pharmacotherapeutics to treat substance use disorders. Several D3R-selective antagonists are effective in animal models of drug abuse, especially in models of relapse. Nevertheless, poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in translating these drug candidates to clinical use. Herein, we report a series of D3R-selective 4-phenylpiperazines with improved metabolic stability. A subset of these compounds was evaluated for D3R functional efficacy and off-target binding at selected 5-HT receptor subtypes, where significant overlap in SAR with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin in wild-type but not D3R knockout mice.

  7. The P2’ residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity

    Energy Technology Data Exchange (ETDEWEB)

    Salameh, M.A.; Soares, A.; Hockla, A.; Radisky, D. C.; Radisky, E. S.

    2011-11-15

    PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumor progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P'{sub 2} position. We find that bulky and charged residues strongly disfavor binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P{sub 1} and P'{sub 2} residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K{sub i} of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.

  8. Tityus gamma toxin, a high affinity effector of the Na+ channel in muscle, with a selectivity for channels in the surface membrane.

    Science.gov (United States)

    Barhanin, J; Ildefonse, M; Rougier, O; Sampaio, S V; Giglio, J R; Lazdunski, M

    1984-01-01

    Toxin gamma from the venom of Tityus serrulatus scorpion produces a partial block of the surface Na+ channel in frog muscle. This block occurs with no change in the voltage-dependence or in the kinetics of the remaining surface Na+ current. The partial blockade of Na+ channel activity occurs with no change in tubular Na+ currents nor in twitch tension. The maximum effect of the toxin is attained at concentrations as low as 3 X 10(-10) M. Hyperpolarization to potentials more negative than the resting potential (E = -90 mV) reduces or abolishes the effect of the toxin. Radioiodinated toxin gamma binds to frog muscle membranes with a very high affinity corresponding to a dissociation constant of about 1 X 10(-11) M. Data obtained with both rabbit and frog muscle indicate that toxin gamma is specific for Na+ channels in surface membranes. Toxin gamma does not seem to bind to Na+ channels in T-tubule membranes. The biochemical data are in good agreement with electrophysiological studies and data on contraction. There is one Tityus gamma toxin binding site per tetrodotoxin binding site in surface membranes. Competition experiments have confirmed that Tityus gamma toxin binds to a new toxin receptor site on the Na+ channel structure. This site is the same that the toxin II from Centruroides suffusus binding site, but this toxin has 100 times less affinity for the Na+ channel than Tityus gamma toxin.

  9. Tityus serrulatus venom contains two classes of toxins. Tityus gamma toxin is a new tool with a very high affinity for studying the Na+ channel.

    Science.gov (United States)

    Barhanin, J; Giglio, J R; Léopold, P; Schmid, A; Sampaio, S V; Lazdunski, M

    1982-11-10

    The interaction of TiTx gamma, the major toxin in the venom of the scorpion Tityus serrulatus, with its receptor in excitable membranes was studied with the use of 125I-TiTx gamma. This derivative retains biological activity, and its specific binding to both brain synaptosomes and electroplaque membranes from Electrophorus electricus is characterized by a dissociation constant equal to that of the native toxin-receptor complex, about 2 to 5 pM. This very high affinity results mainly from a very slow rate of dissociation, equivalent to a half-life longer than 10 h at 4 degrees C. There is a 1:1 stoichiometry between TiTx gamma binding and tetrodotoxin binding to the membranes, but neither tetrodotoxin nor any of 7 other neurotoxins that are representative of 4 different classes of effectors of the Na+ channel interfere with TiTx gamma binding. Similarly, local anesthetics and other molecules that affect other types of ionic channels or neurotransmitter receptors have no effect on TiTx gamma binding. However, toxin II from Centruroides suffusus suffusus does compete with TiTx gamma, though its affinity for the receptor is much lower. Since the Centruroides toxin II is known to affect Na+ channel function, these two scorpion toxins must be put into a fifth class of Na+ channel effectors.

  10. Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

    Science.gov (United States)

    Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi

    2016-07-01

    The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β.

  11. Isolation of a high affinity neutralizing monoclonal antibody against 2009 pandemic H1N1 virus that binds at the 'Sa' antigenic site.

    Directory of Open Access Journals (Sweden)

    Nachiket Shembekar

    Full Text Available Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K(D = 2.1±0.4 pM murine MAb, MA2077 that binds specifically to the hemagglutinin (HA surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC(50 = 0.08 µg/ml. MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 µg/ml against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein 'Sa' and 'Sb' sites were independently mutated, localized the binding site of MA2077 within the 'Sa' antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

  12. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo

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    Gracey Lia E

    2010-07-01

    Full Text Available Abstract Background The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA. Results We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active. This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent. Conclusions These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.

  13. Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications

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    Michelle Cummins

    2014-05-01

    Full Text Available Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA and a ~32 kDa subunit B (RTB. There are fears of ricin being used as a weapon for warfare and terrorism and, as such, there is an increasing need for the development of immunodiagnostic reagents targeted towards this toxin. This article describes the production and characterization of a panel of six ricin-specific monoclonal IgG antibodies (mAbs, previously selected based upon their ability to inhibit ricin-mediated killing of cultured cells. Subsequent epitope binding analysis using the surface plasmon resonance (SPR array biosensor (ProteOn XPR36 indicated three distinct, non-competitive binding epitopes (“bins”. The association (ka and dissociation (kd rate constants and binding affinities (KD of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (KD ranged from 0.1 nM to 9 nM. We present the coding sequences of the variable domains of the six mAbs, the expression, kinetic and cytotoxicity assays for two recombinant Fab (rFab fragments and demonstrate a rFab affinity improvement by chain-shuffling. Together, these antibodies and constituent rFabs represent a panel of reagents for high-affinity recognition of ricin with potential national security biosensor applications.

  14. The solute carrier family 1 (glial high affinity glutamate transporter), member 2 gene, SLC1A2, rs3794087 variant and assessment risk for restless legs syndrome.

    Science.gov (United States)

    Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Martín, Elena; Agúndez, José A G

    2014-02-01

    A glutamatergic dysfunction has been postulated to play a role in restless legs syndrome (RLS) pathophysiology, as glutamate concentrations have been found to increase in the thalamus of RLS patients. The aim of our study was to investigate the possible association between the single nucleotide polymorphism (SNP) rs3794087 in the solute carrier family 1 (glial high affinity glutamate transporter), member 2 gene, SLC1A2, related with glutamate transport and the risk for RLS. We studied the allelic and genotype frequencies of the SNP rs3794087 in 205 patients with RLS and 328 healthy controls using TaqMan genotyping. The rs3794087 genotype and allelic frequencies did not significantly differ between patients with RLS and controls and were unrelated with the age at onset of RLS, gender, and family history of RLS. The results of our study suggest that the rs3794087 polymorphism is not related to the risk for RLS. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Elution of High-affinity (>10-9 KD) Antibodies from Tissue Sections: Clues to the Molecular Mechanism and Use in Sequential Immunostaining.

    Science.gov (United States)

    Gendusa, Rossella; Scalia, Carla Rossana; Buscone, Serena; Cattoretti, Giorgio

    2014-07-01

    Inconsistent results obtained with published methods for the elution of antibodies from tissue sections prompted the assessment of both old and new methods in combination with monoclonal rabbit antibodies of known, increased affinity (above 1×10(-9) KD). We tested an acidic (pH 2) glycine buffer, a 6 M urea hot buffer and a 2-Mercaptoethanol, SDS buffer (2-ME/SDS). Some antibodies were not removed by the glycine pH 2 or 6 M urea hot buffers, indicating that antibodies survive much harsher conditions than previously believed. We found that the elution is dependent upon the antibody affinity and is reduced by species-specific crosslinking via a dimeric or Fab fragments of a secondary antibody. The high affinity bond of exogenous streptavidin with the endogenous biotin can be removed by 6 M urea but not by the other buffers. 2-ME/SDS buffer is superior to glycine pH 2 and 6 M urea hot elution buffers for all antibodies because of its irreversible effect on the structure of the antibodies. It also has a mild retrieving effect on some antigens present on routinely treated sections and no detrimental effect on the immunoreactivity of the tissue. Therefore, 2-ME/SDS buffer is the method of choice to perform multiple rounds of immunostaining on a single routine section.

  16. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  17. High Throughput Screening of High-Affinity Ligands for Proteins with Anion-Binding Sites using Desorption Electrospray Ionization (DESI) Mass Spectrometry

    Science.gov (United States)

    Lu, Xin; Ning, Baoming; He, Dacheng; Huang, Lingyun; Yue, Xiangjun; Zhang, Qiming; Huang, Haiwei; Liu, Yang; He, Lan; Ouyang, Jin

    2014-03-01

    A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

  18. Vsx2 controls eye organogenesis and retinal progenitor identity via homeodomain and non-homeodomain residues required for high affinity DNA binding.

    Directory of Open Access Journals (Sweden)

    Changjiang Zou

    2012-09-01

    Full Text Available The homeodomain and adjacent CVC domain in the visual system homeobox (VSX proteins are conserved from nematodes to humans. Humans with missense mutations in these regions of VSX2 have microphthalmia, suggesting both regions are critical for function. To assess this, we generated the corresponding mutations in mouse Vsx2. The homeodomain mutant protein lacked DNA binding activity and the knock-in mutant phenocopied the null mutant, ocular retardation J. The CVC mutant protein exhibited weakened DNA binding; and, although the corresponding knock-in allele was recessive, it unexpectedly caused the strongest phenotype, as indicated by severe microphthalmia and hyperpigmentation of the neural retina. This occurred through a cryptic transcriptional feedback loop involving the transcription factors Mitf and Otx1 and the Cdk inhibitor p27(Kip1. Our data suggest that the phenotypic severity of the CVC mutant depends on the weakened DNA binding activity elicited by the CVC mutation and a previously unknown protein interaction between Vsx2 and its regulatory target Mitf. Our data also suggest that an essential function of the CVC domain is to assist the homeodomain in high-affinity DNA binding, which is required for eye organogenesis and unhindered execution of the retinal progenitor program in mammals. Finally, the genetic and phenotypic behaviors of the CVC mutation suggest it has the characteristics of a recessive neomorph, a rare type of genetic allele.

  19. The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.

    Science.gov (United States)

    Laver, Derek R; Hamada, Tomoyo; Fessenden, James D; Ikemoto, Noriaki

    2007-12-01

    In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

  20. Calcium paradox and calcium entry blockers

    NARCIS (Netherlands)

    Ruigrok, T.J.C.; Slade, A.M.; Nayler, W.G.; Meijler, F.L.

    1984-01-01

    Reperfusion of isolated hearts with calcium-containing solution after a short period of calcium-free perfusion results in irreversible cell damage (calcium paradox). This phenomenon is characterized by an excessive influx of calcium into the cells, the rapid onset of myocardial contracture, exhausti