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Sample records for high-affinity l-ascorbic acid

  1. The preparation and the sustained release of titanium dioxide hollow particles encapsulating L-ascorbic acid

    Science.gov (United States)

    Tominaga, Yoko; Kadota, Kazunori; Shimosaka, Atsuko; Yoshida, Mikio; Oshima, Kotaro; Shirakawa, Yoshiyuki

    2018-05-01

    The preparation of the titanium dioxide hollow particles encapsulating L-ascorbic acid via sol-gel process using inkjet nozzle has been performed, and the sustained release and the effect protecting against degradation of L-ascorbic acid in the particles were investigated. The morphology of titanium dioxide particles was evaluated by scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). The sustained release and the effect protecting against degradation of L-ascorbic acid were estimated by dialysis bag method in phosphate buffer saline (PBS) (pH = 7.4) as release media. The prepared titanium dioxide particles exhibited spherical porous structures. The particle size distribution of the titanium dioxide particles was uniform. The hollow titanium dioxide particles encapsulating L-ascorbic acid showed the sustained release. It was also found that the degradation of L-ascorbic acid could be inhibited by encapsulating L-ascorbic acid in the titanium dioxide hollow particles.

  2. Metabolic Conversion of l-Ascorbic Acid to Oxalic Acid in Oxalate-accumulating Plants 1

    Science.gov (United States)

    Yang, Joan C.; Loewus, Frank A.

    1975-01-01

    l-Ascorbic acid-1-14C and its oxidation product, dehydro-l-ascorbic acid, produced labeled oxalic acid in oxalate-accumulating plants such as spinach seedlings (Spinacia oleracea) and the detached leaves of woodsorrel (Oxalis stricta and O. oregana), shamrock (Oxalis adenopylla), and begonia (Begonia evansiana). In O. oregana, conversion occurred equally well in the presence or absence of light. This relationship between l-ascorbic acid metabolism and oxalic acid formation must be given careful consideration in attempts to explain oxalic accumulation in plants. PMID:16659288

  3. Structural Requirements of Alkylglyceryl-l-Ascorbic Acid Derivatives for Melanogenesis Inhibitory Activity.

    Science.gov (United States)

    Taira, Norihisa; Katsuyama, Yushi; Yoshioka, Masato; Muraoka, Osamu; Morikawa, Toshio

    2018-04-10

    l-Ascorbic acid has multifunctional benefits on skin aesthetics, including inhibition of melanin production, and is widely used in cosmetics. It, however, has low stability and poor skin penetration. We hypothesize that alkylglyceryl-l-ascorbic acid derivatives, highly stable vitamin C-alkylglycerol conjugates, would have similar anti-melanogenic activity with better stability and penetration. We test 28 alkylglyceryl-l-ascorbic acid derivatives ( 1 - 28 ) on theophylline-stimulated B16 melanoma 4A5 cells to determine if they inhibit melanogenesis and establish any structure-function relationships. Although not the most potent inhibitors, 3- O -(2,3-dihydroxypropyl)-2- O -hexyl-l-ascorbic acid ( 6 , IC 50 = 81.4 µM) and 2- O -(2,3-dihydroxypropyl)-3- O -hexyl-l-ascorbic acid ( 20 , IC 50 = 117 µM) are deemed the best candidate derivatives based on their inhibitory activities and low toxicities. These derivatives are also found to be more stable than l-ascorbic acid and to have favorable characteristics for skin penetration. The following structural requirements for inhibitory activity of alkylglyceryl-l-ascorbic acid derivatives are also determined: (i) alkylation of glyceryl-l-ascorbic acid is essential for inhibitory activity; (ii) the 3- O -alkyl-derivatives ( 2 - 14 ) exhibit stronger inhibitory activity than the corresponding 2- O -alkyl-derivatives ( 16 - 28 ); and (iii) derivatives with longer alkyl chains have stronger inhibitory activities. Mechanistically, our studies suggest that l-ascorbic acid derivatives exert their effects by suppressing the mRNA expression of tyrosinase and tyrosine-related protein-1.

  4. Synthesis of L-ascorbic acid in the phloem

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    Haupt Sophie

    2003-11-01

    Full Text Available Abstract Background Although plants are the main source of vitamin C in the human diet, we still have a limited understanding of how plants synthesise L-ascorbic acid (AsA and what regulates its concentration in different plant tissues. In particular, the enormous variability in the vitamin C content of storage organs from different plants remains unexplained. Possible sources of AsA in plant storage organs include in situ synthesis and long-distance transport of AsA synthesised in other tissues via the phloem. In this paper we examine a third possibility, that of synthesis within the phloem. Results We provide evidence for the presence of AsA in the phloem sap of a wide range of crop species using aphid stylectomy and histochemical approaches. The activity of almost all the enzymes of the primary AsA biosynthetic pathway were detected in phloem-rich vascular exudates from Cucurbita pepo fruits and AsA biosynthesis was demonstrated in isolated phloem strands from Apium graveolens petioles incubated with a range of precursors (D-glucose, D-mannose, L-galactose and L-galactono-1,4-lactone. Phloem uptake of D-[U-14C]mannose and L-[1-14C]galactose (intermediates of the AsA biosynthetic pathway as well as L-[1-14C]AsA and L-[1-14C]DHA, was observed in Nicotiana benthamiana leaf discs. Conclusions We present the novel finding that active AsA biosynthesis occurs in the phloem. This process must now be considered in the context of mechanisms implicated in whole plant AsA distribution. This work should provoke studies aimed at elucidation of the in vivo substrates for phloem AsA biosynthesis and its contribution to AsA accumulation in plant storage organs.

  5. The browning kinetics of the non-enzymatic browning reaction in L-ascorbic acid/basic amino acid systems

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    Ai-Nong YU

    Full Text Available Abstract Under the conditions of weak basis and the reaction temperature range of 110-150 °C, lysine, arginine and histidine were reacted with L-ascorbic acid at equal amount for 30-150 min, respectively and the formation of browning products was monitored with UV–vis spectrometry. The kinetic characteristics of their non-enzymatic browning reaction were investigated. The study results indicated that the non-enzymatic browning reaction of these three amino acids with L-ascorbic acid to form browning products was zero-order reaction. The apparent activation energies for the formation of browning products from L-ascorbic acid/lysine, L-ascorbic acid/arginine and L-ascorbic acid/histidine systems were 54.94, 50.08 and 35.31kJ/mol. The activation energy data indicated the degree of effects of reaction temperature on non-enzymatic browning reaction. Within the temperature range of 110-150 °C, the reaction rate of L-ascorbic acid/lysine system was the fastest one, followed by that of the L-ascorbic acid/arginine system. The reaction rate of L-ascorbic acid/histidine system was the slowest one. Based on the observed kinetic data, the formation mechanisms of browning products were proposed.

  6. Neutron scattering and HPLC study on L-ascorbic acid and its degradation

    Energy Technology Data Exchange (ETDEWEB)

    Bellocco, E. [Department of Organic and Biological Chemistry, University of Messina, Messina (Italy)], E-mail: bellocco@isengard.unime.it; Barreca, D.; Lagana, G.; Leuzzi, U. [Department of Organic and Biological Chemistry, University of Messina, Messina (Italy); Migliardo, F.; Torre, R. La; Galli, G. [Department of Physics, University of Messina, Messina (Italy); Galtieri, A. [Department of Organic and Biological Chemistry, University of Messina, Messina (Italy); Minutoli, L.; Squadrito, F. [Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina (Italy)

    2008-04-18

    The present paper shows a systematic dynamic and kinetic study on L-ascorbic acid and its degradation at high temperature. The neutron scattering study allows, through the behavior of quasi-elastic neutron scattering (QENS) spectra, to characterize the diffusive dynamics of L-ascorbic acid in water mixtures. Ascorbic acid undergoes degradation process at high temperature, but the presence of trehalose in solution markedly avoids ascorbic acid loss enhancing its t{sub 1/2} (half life time), as determined by high performance liquid chromatography (HPLC)

  7. Neutron scattering and HPLC study on L-ascorbic acid and its degradation

    International Nuclear Information System (INIS)

    Bellocco, E.; Barreca, D.; Lagana, G.; Leuzzi, U.; Migliardo, F.; Torre, R. La; Galli, G.; Galtieri, A.; Minutoli, L.; Squadrito, F.

    2008-01-01

    The present paper shows a systematic dynamic and kinetic study on L-ascorbic acid and its degradation at high temperature. The neutron scattering study allows, through the behavior of quasi-elastic neutron scattering (QENS) spectra, to characterize the diffusive dynamics of L-ascorbic acid in water mixtures. Ascorbic acid undergoes degradation process at high temperature, but the presence of trehalose in solution markedly avoids ascorbic acid loss enhancing its t 1/2 (half life time), as determined by high performance liquid chromatography (HPLC)

  8. The comparison of techniques and methods for L-ascorbic acid determination in the fruits

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    Cvetković Biljana R.

    2012-01-01

    Full Text Available Vitamin C is an essential vitamin for human nutrition; with the L-ascorbic acid (AA being the active form of vitamin C. Hence, determination of the L-ascorbic acid in the natural and processed foods is very important. In the past, plenty of methods based on the reversible redox reaction of AA oxidation/DHA reduction were developed. Because of L-ascorbic acid instability in aqueous solutions, it is useful to analyze various types of extraction. The aim of this study is to compare three different methods and three different extractants for the L-ascorbic acid determination. Fruits (kiwi, lemon, orange, and grapefruit were purchased from a local market. The L-ascorbic acid in these four samples was determined by the three different methods: the AOAC, the HPLC method with three different types of extractions, and the colorimetric method using ascorbate-oxidase. For the HPLC measurements, one part of the fruits was extracted with distilled water, the second with potassium hydrogen phosphate, and the third with 3% meta-phosphoric acid (MPA in 8% acetic acid. The HPLC measurements of each sample were repeated three times, the AOAC titration was repeated five times, and in the calorimetric method three measurements were performed. The results were statistically evaluated related to sample basis. Statistical analysis shows that there is a significant difference between the results for all three methods of extraction for all samples, except for the grapefruit sample where no significant difference was observed between the results obtained after the buffer extraction (E2 and the metaphosphoric acid in acetic acid extraction (E3. Discriminative analysis for the HPLC determinations proves that there is a clear difference and defined border between the samples in relation to the methods of extraction during the HPLC determination.

  9. [Nickel exposure to A549 cell damage and L-ascorbic acid interference effect].

    Science.gov (United States)

    Fu, Yao; Wang, Yue; Dan, Han; Zhang, Lin; Ma, Wenhan; Pan, Yulin; Wu, Yonghui

    2015-05-01

    Studying different concentrations of nickel smelting smoke subjects of human lung adenocarcinoma cells (A549) carcinogenic effects, discusses the influence of L-ascorbic acid protection. The A549 cells were divided into experimental and L-ascorbic acid in the intervention group. Plus exposure group concentration of nickel refining dusts were formulated 0.00, 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml suspension, the intervention group on the basis of the added exposure group containing L-ascorbic acid (100 mmol/L), contact 24 h. Detection of cell viability by MTT assay. When the test substance concentration select 0.00, 25.00, 50.00, 100.00 µg/ml experiment for internal Flou-3 fluorescent probe to detect cell Ca²⁺ concentration, within DCFH-DA detect intracellular reactive oxygen (ROS) content, real-time quantitative PCR (real time, in the RT-PCR) was used to detect cell HIF-1α gene expression. With the increase of concentration, subjects increased cell growth inhibition rate, intracellular Ca²⁺ concentration increases, ROS content increased, HIF-1α gene expression increased, differences were statistically significant (P nickel exposure damage to cells. With subjects following exposure to nickel concentration increased, its effect on A549 cell damage increases, L-ascorbic acid cell damage caused by nickel has certain protective effect.

  10. Neurobehavioural and Neurotoxic Effects of L-ascorbic Acid and L ...

    African Journals Online (AJOL)

    Background: Lead is an environmental toxicant, occupational and environmental exposures remain a serious problem in developing and industrializing countries. Objective: This study is designed to investigate the effects of L-ascorbic acid and L-tryptophan on the neurotoxicity and neurobehavioural alterations in lead ...

  11. Effects of L-ascorbic acid and alpha-tocopherol on biochemical ...

    African Journals Online (AJOL)

    Background: The purpose of this study is to determine the effect of L-ascorbic acid and alpha-tocopherol as well as combination of these vitamins with or without exposure to physical exercise on intensity of lipid peroxidation, activity of xanthine oxidase, activity of total antioxidative system, concentration of glutathione, and ...

  12. Clinical efficacy of 25% L-ascorbic acid (C'ensil) in the treatment of melasma.

    Science.gov (United States)

    Hwang, Seon-Wook; Oh, Doo-Jin; Lee, Deborah; Kim, Jung-Wook; Park, Sung-Wook

    2009-01-01

    L-Ascorbic acid is used to treat melasma; however, it is quickly oxidized in aqueous solutions. Thus, C'ensil, a formulation containing 25% l-ascorbic acid and a chemical penetration enhancer, was created to promote the penetration of l-ascorbic acid into the skin. To evaluate the efficacy of C'ensil in patients with melasma. Forty subjects with melasma were treated with C'ensil during an open-label trial over a period of 16 weeks. Each subject's skin pigmentation was assessed every 4 weeks using the Melasma Area and Severity Index (MASI) and mexameter score. In addition, transepidermal water loss, skin dryness and irritation, and quality of life (Melasma Quality of Life Scale [MelasQoL]) were evaluated. After 16 weeks, a significant decrease was noted in the degree of pigmentation based on the patients' MASI and mexameter scores. MelasQoL scores also decreased, indicating an increase in the subjects' quality of life. Our data indicate that C'ensil is an effective treatment modality for melasma.

  13. Solubilities of magnesium-L-ascorbate, calcium-L-ascorbate, magnesium-L-glutamate, magnesium-D-gluconate, calcium-D-gluconate, calcium-D-heptagluconate, L-aspartic acid, and 3-nitrobenzoic acid in water

    Energy Technology Data Exchange (ETDEWEB)

    Mishelevich, Alexander [Department of Chemical Engineering, Ben Gurion University of the Negev, Beer Sheva 84105 (Israel); Apelblat, Alexander [Department of Chemical Engineering, Ben Gurion University of the Negev, Beer Sheva 84105 (Israel)], E-mail: apelblat@bgu.ac.il

    2008-05-15

    The solubility in water of magnesium-L-ascorbate, calcium-L-ascorbate, magnesium-L-glutamate, magnesium-D-gluconate, calcium-D-gluconate, calcium-D-heptagluconate, L-aspartic acid, and 3-nitrobenzoic acid was determined in the 278.15 K to 343.15 K temperature range. The solubility of these compounds served to permit the evaluation of the apparent molar enthalpies of solution.

  14. Solubilities of magnesium-L-ascorbate, calcium-L-ascorbate, magnesium-L-glutamate, magnesium-D-gluconate, calcium-D-gluconate, calcium-D-heptagluconate, L-aspartic acid, and 3-nitrobenzoic acid in water

    International Nuclear Information System (INIS)

    Mishelevich, Alexander; Apelblat, Alexander

    2008-01-01

    The solubility in water of magnesium-L-ascorbate, calcium-L-ascorbate, magnesium-L-glutamate, magnesium-D-gluconate, calcium-D-gluconate, calcium-D-heptagluconate, L-aspartic acid, and 3-nitrobenzoic acid was determined in the 278.15 K to 343.15 K temperature range. The solubility of these compounds served to permit the evaluation of the apparent molar enthalpies of solution

  15. An efficient synthesis of tetramic acid derivatives with extended conjugation from L-Ascorbic Acid

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    Bisht Surendra S

    2006-12-01

    Full Text Available Abstract Background Tetramic acids with polyenyl substituents are an important class of compounds in medicinal chemistry. Both solid and solution phase syntheses of such molecules have been reported recently. Thiolactomycin, a clinical candidate for treatment of tuberculosis has led to further explorations in this class. We have recently developed an efficient synthesis of tetramic acids derivatives from L- ascorbic acid. In continuation of this work, we have synthesised dienyl tetramic acid derivatives. Results 5,6-O-Isopropylidene-ascorbic acid on reaction with DBU led to the formation of tetronolactonyl allyl alcohol, which on oxidation with pyridinium chlorochromate gave the respective tetranolactonyl allylic aldehydes. Wittig olefination followed by reaction of the resulting tetranolactonyl dienyl esters with different amines resulted in the respective 5-hydroxy lactams. Subsequent dehydration of the hydroxy lactams with p-toluene sulphonic acid afforded the dienyl tetramic acid derivatives. All reactions were performed at ambient temperature and the yields are good. Conclusion An efficient and practical method for the synthesis of dienyl tetramic acid derivatives from inexpensive and easily accessible ascorbic acid has been developed. The compounds bear structural similarities to the tetramic acid based polyenic antibiotics and thus this method offers a new and short route for the synthesis of tetramic acid derivatives of biological significance.

  16. Skin penetration and retention of L-ascorbic acid 2-phosphate using multilamellar vesicles.

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    Yoo, Juno; Shanmugam, Srinivasan; Song, Chung-Kil; Kim, Dae-Duk; Choi, Han-Gon; Yong, Chul-Soon; Woo, Jong-Soo; Yoo, Bong Kyu

    2008-12-01

    Transdermal formulation of L-ascorbic acid 2-phosphate magnesium salt (A2P) was prepared using multilamellar vesicles (MLV). A2P was either physically mixed with or entrapped into three different MLVs of neutral, cationic, and anionic liposome vesicles. For the preparation of neutral MLVs, phosphatidylcholine (PC) and cholesterol (CH) were used. For cationic and anionic MLVs, dioleoyl-trimethylammonium-propane and dimyristoyl glycerophosphate were added as surface charge inducers, respectively, in addition to PC and CH. Particle size of the three A2P-loaded MLVs was submicron, and polydispersity index revealed homogenous distribution of the prepared MLVs except neutral ones. Skin penetration study with hairless mouse skin showed that both physical mixtures of A2P with empty MLVs and A2P-loaded MLVs increased penetration of the drug compared to aqueous A2P solution. During the penetration, however, significant amount of the drug was metabolized into L-ascorbic acid, which has no beneficial effect on stimulation of hair growth. Out of the physical mixtures and A2P-loaded MLVs tested, physical mixture of A2P with empty cationic MLV resulted in the greatest skin penetration and retention in hairless mouse skin.

  17. Effect of L-ascorbic acid on nickel-induced alterations in serum lipid profiles and liver histopathology in rats.

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    Das, Kusal K; Gupta, Amrita Das; Dhundasi, Salim A; Patil, Ashok M; Das, Swastika N; Ambekar, Jeevan G

    2006-01-01

    Nickel exposure greatly depletes intracellular ascorbate and alters ascorbate-cholesterol metabolism. We studied the effect of the simultaneous oral treatment with L-ascorbic acid (50 mg/100 g body weight (BW) and nickel sulfate (2.0 mg/100 g BW, i.p) on nickelinduced changes in serum lipid profiles and liver histopathology. Nickel-treated rats showed a significant increase in serum low-density lipoprotein-cholesterol, total cholesterol, triglycerides, and a significant decrease in serum high-density lipoprotein-cholesterol. In the liver, nickel sulfate caused a loss of normal architecture, fatty changes, extensive vacuolization in hepatocytes, eccentric nuclei, and Kupffer cell hypertrophy. Simultaneous administration of L-ascorbic acid with nickel sulfate improved both the lipid profile and liver impairments when compared with rats receiving nickel sulfate only. The results indicate that L-ascorbic acid is beneficial in preventing nickel-induced lipid alterations and hepatocellular damage.

  18. The use of L-ascorbic acid in speciation of arsenic compounds in drinking water

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    Marjanović Nikola J.

    2009-01-01

    Full Text Available Arsenic speciation, besides total arsenic content determination, is very important in analysis of water, foodstuffs, and environmental samples, because of varying degrees of toxicity of different species. For such purpose hydride generation atomic absorption spectrometry can be used based on the generation of certain types of hydride, depending on the pH value and pretreatment in different reaction media. In this study, we have investigated the effect of L-ascorbic acid as the reaction medium as well as the pre-reducing agent in speciation of arsenic by hydride generation-atomic absorption spectrometry in order to determine monomethyl arsonic acid (MMA in the presence of inorganic forms of arsenic.

  19. Effect of solvent on anodization of Zr-4 in L-ascorbic acid - SEM studies

    International Nuclear Information System (INIS)

    Jeevana Jyothi, V.; Ch Anjaneyulu

    2013-01-01

    Anodization of Zircaloy-4 in 0.1 M L-ascorbic acid has been carried out. Kinetics of anodic oxidation of Zircaloy-4 has been studied at a constant current density of 8 mA.cm -2 and at room temperature. The plots of formation voltage vs time, reciprocal capacitance vs time and reciprocal capacitance vs formation voltage were drawn. From these plots, formation rate, current efficiency and differential field were calculated. The addition of solvent (ethylene glycol) showed better kinetic results for 20, 40, 60 and 80% aquo-glycolic media, the dielectric constant values are low leading to the marked improvement in the kinetics. The surface morphology of the anodic films was also studied by Scanning Electron Micrographs (SEM). (author)

  20. Dielectric behaviour and intermolecular association between L(+) ascorbic acid and ethanol

    International Nuclear Information System (INIS)

    Rudyk, R.A.; Torres, M.C.; Acuna Molina, M.A.

    1990-01-01

    In order to determine the dipole moment of L(+) ascorbic acid and the relation to its structure the experimental variations of permitivities, refractive indices and specific volumes of a series of dilute ethanolic solutions at 25 deg C were examined. The average moment (μ) using Buckingham equation was found to be 5,58 D considering the spherical approximation and 7,81 D if the ellipsoidal form factor was considered. The calculated μ value through vectorial addition was 4,98 D. The solute partial molal volume in the studied range was calculated to be 94,73 cm 3 instead of the theoretical value of 106,71 cm 3 . Both discrepancies are attributed to intermolecular solute-solvent interactions. A possible electronic displacement which favours hydrogen bonding with the solvent is postulated. (Author) [es

  1. Mechanism of degradation and discoloration reaction of L-ascorbic acid

    International Nuclear Information System (INIS)

    Kurata, Tadao

    1976-01-01

    The mechanisms of decomposition and coloration reaction of L-ascorbic acid are reviewed. At the initial stage of the decomposition, it can be classified roughly into oxidative and non-oxidative processes of decomposition. ASA forms furfural by being heated and decomposed in strong acid. The mechanism of the production of furfural at varying pH in acidic region was discussed. Furfural was produced through the enol form of 3-deoxy-L-pentosulose(3DP). 3DP seemed to be produced by two different routes: the one route consists of successive reactions from ASA through lactone ring-opening, dehydration, decarboxylation, to 3DP, and the other consists of reactions from the 3-keto form of ASA, through lactone ring-opening, decarboxylation, and dehydration, to the enol form of 3DP. ASA is easily reduced and decomposed through dehydro-ASA(DHA) by the presence of an oxidizing agent. The decomposition of DHA is discussed in cases of the systems of DHA alone, DHA and α-aminoacid, and DHA and amine. DHA was decomposed by the same reaction scheme as the decomposition of ASA and yielded 2-furoic acid. In the presence of an amino acid, DHA was decomposed by the Strecker decomposition, and yielded a red compound and a radical. In the presence of an amine, the discoloration reaction seemed to take place through radical reaction mechanism. The coloration reaction of ASA occurs in an acidic medium, and is accelerated by the oxidative process of decomposition. (Nishino, S.)

  2. The role of topically applied L-ascorbic acid in ex-vivo examination of burn-injured human skin

    Science.gov (United States)

    Pielesz, Anna; Biniaś, Dorota; Bobiński, Rafał; Sarna, Ewa; Paluch, Jadwiga; Waksmańska, Wioletta

    2017-10-01

    Wound treatment and healing is complex and is comprised of an elaborate set of processes including cellular, spectroscopic and biochemical ones as well as the ;reaction; of local tissue to thermal injury. Vitamin C as L-ascorbic acid (LA) prevents injurious effects of oxidants because it reduces reactive oxygen species to stable molecules, it becomes oxidized to the short-lived ascorbyl radical. As a result, antioxidant treatment may contribute to minimizing injury in burn patients. The aim of this study is to assess changes in molecular structure of collagen extracted from human epidermis burn wound scab during incubation of the epidermis in L-ascorbic acid solution. The study will be performed using FTIR and FT Raman spectroscopies. During this research it was observed that the intensity of Raman peaks increased where healing was being modified by LA. The intensity of the amide III band at 1247 cm- 1 relative to the intensity at 1326 cm- 1 was used to test tissue repair degree at the incision site. FTIR spectra were recorded from frozen specimens of serum modified by LA; an analysis of shifts in the amide I band position was conducted. The appearance of a new band for frozen samples modified by LA was observed around 1149-1220 cm- 1. The above conclusions confirmed the creation of hydrogen bonds between Nsbnd H stretch and Cdbnd O. Samples being incubated in solutions of L-ascorbic acid demonstrated the absence of electrophoretic bands of albumin. Alterations in the surface of the skin incubated in L-ascorbic acid were investigated with the use of Scanning Electron Microscopy (SEM). A decrease in external symptoms of burn injury was noted in the damaged epidermis incubated in L-ascorbic acid.

  3. Investigation of L(+)-Ascorbic Acid with Raman Spectroscopy in Visible and UV Light

    DEFF Research Database (Denmark)

    Berg, Rolf W.

    2015-01-01

    Abstract: Raman spectroscopy investigations of l(+)-ascorbic acid and its mono- and di-deprotonated anions (AH(-) and A(2-)) are reviewed and new measurements reported with several wavelengths, 229, 244, 266, 488, and 532nm. Results are interpreted, assisted by new DFT/B3LYP quantum chemical calc......). Relatively weak preresonance enhancement was seen for A(2-) when excitation was done with 229nm UV light, allowing water bands to become observable as for normal visible light Raman spectra....... calculations with 6-311++G(d,p) basis sets for several conformations of ascorbic acid and the anions. Raman spectra were measured during titration with NaOH base in an oxygen-poor environment to avoid fluorescence when solutions were alkaline. The ultraviolet (UV) absorption band for ascorbic acid in aqueous......cm(-1). Finally, for the ascorbate di-anion, absorption was found at similar to 298.4nm with molar absorptivity of similar to 7,000 L mol(-1) cm(-1) and below similar to 220nm. With UV light (244 and 266nm), strongly basic solutions gave pronounced Raman resonance enhancement at similar to 1556cm(-1...

  4. Modelling the effects of transglutaminase and L-ascorbic acid on substandard quality wheat flour by response surface methodology

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    Šimurina Olivera D.

    2014-01-01

    Full Text Available In recent decade, there have been observed extreme variations in climatic conditions which in combination with inadequate agro techniques lead to decreased quality of mercantile wheat, actally flour. The application of improvers can optimise the quality of substandard wheat flour. This paper focuses to systematic analysis of individual and interaction effects of ascorbic acid and transglutaminase as dough strengthening improvers. The effects were investigated using the Response Surface Methodology. Transglutaminase had much higher linear effect on the rheological and fermentative properties of dough from substandard flour than L-ascorbic acid. Both transglutaminase and L-ascorbic acid additions had a significant linear effect on the increase of bread specific volume. Effects of transglutaminase and ascorbic acid are dependent on the applied concentrations and it is necessary to determine the optimal concentration in order to achieve the maximum quality of the dough and bread. Optimal levels of tested improvers were determined using appropriate statistical techniques which applied the desirability function. It was found that the combination of 30 mg/kg of transglutaminase and 75.8 mg/kg of L-ascorbic acid achieved positive synergistic effect on rheological and fermentative wheat dough properties, as well on textural properties and specific volume of bread made from substandard quality flour.

  5. Effect of high doses of L-ascorbic acid on the antioxidative/oxidative state in the rats

    DEFF Research Database (Denmark)

    Niemiec, T.; Sawosz, E.; Chwalibog, André

    2005-01-01

    The objective of this study was to determine the effects of mega-doses of vitamin C (0.3, 0.6 and 0.9% of diet) as a dietary supplement for rats on selected indices of the antioxidative/oxidative state in 40 growing Wistar rats (4x10). It was found that L-ascorbic acid and Total Antioxidative State...... (TAS) in plasma did not increase with increasing vitamin C supply. The results indicate that high doses of L-ascorbic acid (0.3 and 0.9 but not 0.6%) increased the concentration of this antioxidant in plasma. Supplementation of vitamin C above 0.3% to the diets had pro-oxidative effects on lipid...... structures, while application of 0.9% promoted oxidative degradation of rat livers....

  6. An evaluation of the antiaging properties of strawberry hydrolysate treatment enriched with L-ascorbic acid applied with microneedle mesotherapy.

    Science.gov (United States)

    Markiewicz, Agata; Zasada, Malwina; Erkiert-Polguj, Anna; Wieckowska-Szakiel, Marzena; Budzisz, Elzbieta

    2018-04-16

    Mature skin is characterized by a loss of elasticity, hyperpigmentation, and dehydration. L-ascorbic acid stimulates the synthesis of collagen type I, inhibits melanogenesis, and helps to maintain correct skin hydration. Combining microneedle mesotherapy with the application of preparations rich in vitamin C results in better therapeutic effects due to the improved absorption of active substances. The study evaluates the effectiveness of the application of strawberry hydrolysate enriched with L-ascorbic acid using microneedle mesotherapy. Seventeen volunteers aged 45-70 years underwent a series of four microneedle mesotherapy treatments with vitamin C serum, performed every 10 days. The 20% L-ascorbic acid solution (pH = 3.5) was prepared immediately before application. After the treatment, the participants gave a subjective assessment of the effectiveness. Cutometer ® was used to measure skin elasticity and firmness, Corneometer ® to measure skin hydration, and Mexameter ® skin tone. The results of the survey showed improvements in skin hydration and elasticity. In vivo studies confirmed the effectiveness of serum and the impact of the active substance on skin firmness and elasticity, the degree of hydration and skin tone. Microneedling with vitamin C improves skin tone, hydratation and firmness, and decreases the visibility of hyperpigmentation. © 2018 Wiley Periodicals, Inc.

  7. The Rheological Properties of Lipid Monolayers Modulate the Incorporation of l-Ascorbic Acid Alkyl Esters.

    Science.gov (United States)

    Díaz, Yenisleidy de Las Mercedes Zulueta; Mottola, Milagro; Vico, Raquel V; Wilke, Natalia; Fanani, María Laura

    2016-01-19

    In this work, we tested the hypothesis that the incorporation of amphiphilic drugs into lipid membranes may be regulated by their rheological properties. For this purpose, two members of the l-ascorbic acid alkyl esters family (ASCn) were selected, ASC16 and ASC14, which have different rheological properties when organized at the air/water interface. They are lipophilic forms of vitamin C used in topical pharmacological preparations. The effect of the phase state of the host lipid membranes on ASCn incorporation was explored using Langmuir monolayers. Films of pure lipids with known phase states have been selected, showing liquid-expanded, liquid-condensed, and solid phases as well as pure cholesterol films in liquid-ordered state. We also tested ternary and quaternary mixed films that mimic the properties of cholesterol containing membranes and of the stratum corneum. The compressibility and shear properties of those monolayers were assessed in order to define its phase character. We found that the length of the acyl chain of the ASCn compounds induces differential changes in the rheological properties of the host membrane and subtly regulates the kinetics and extent of the penetration process. The capacity for ASCn uptake was found to depend on the phase state of the host film. The increase in surface pressure resultant after amphiphile incorporation appears to be a function of the capacity of the host membrane to incorporate such amphiphile as well as the rheological response of the film. Hence, monolayers that show a solid phase state responded with a larger surface pressure increase to the incorporation of a comparable amount of amphiphile than liquid-expanded ones. The cholesterol-containing films, including the mixture that mimics stratum corneum, allowed a very scarce ASCn uptake independently of the membrane diffusional properties. This suggests an important contribution of Cho on the maintenance of the barrier function of stratum corneum.

  8. The effects of reactants ratios, reaction temperatures and times on Maillard reaction products of the L-ascorbic acid/L-glutamic acid system

    Directory of Open Access Journals (Sweden)

    Yong-Yan ZHOU

    2016-01-01

    Full Text Available Abstract The transformation law of the Maillard reaction products with three different reactants ratios - equimolar reactants, excess L-glutamic acid and excess L-ascorbic acid reaction respectively, five different temperatures, and different time conditions for the L-ascorbic acid / L-glutamic acid system were investigated. Results showed that, the increase of the reaction time and temperature led to the increase of the browning products, uncoloured intermediate products, as well as aroma compounds. Compared with the equimolar reaction system, the excess L-ascorbic acid reaction system produced more browning products and uncoloured intermediate products, while the aroma compounds production remained the same. In the excess L-glutamic acid system, the uncoloured intermediate products increased slightly, the browning products remained the same, while the aroma compounds increased.

  9. Thermal stability of L-ascorbic acid and ascorbic acid oxidase in broccoli (Brassica oleracea var. italica).

    Science.gov (United States)

    Munyaka, Ann Wambui; Makule, Edna Edward; Oey, Indrawati; Van Loey, Ann; Hendrickx, Marc

    2010-05-01

    The thermal stability of vitamin C (including l-ascorbic acid [l-AA] and dehydroascorbic acid [DHAA]) in crushed broccoli was evaluated in the temperature range of 30 to 90 degrees C whereas that of ascorbic acid oxidase (AAO) was evaluated in the temperature range of 20 to 95 degrees C. Thermal treatments (for 15 min) of crushed broccoli at 30 to 60 degrees C resulted in conversion of l-AA to DHAA whereas treatments at 70 to 90 degrees C retained vitamin C as l-AA. These observations indicated that enzymes (for example, AAO) could play a major role in the initial phase (that is, oxidation of l-AA to DHAA) of vitamin C degradation in broccoli. Consequently, a study to evaluate the temperature-time conditions that could result in AAO inactivation in broccoli was carried out. In this study, higher AAO activity was observed in broccoli florets than stalks. During thermal treatments for 10 min, AAO in broccoli florets and stalks was stable until around 50 degrees C. A 10-min thermal treatment at 80 degrees C almost completely inactivated AAO in broccoli. AAO inactivation followed 1st order kinetics in the temperature range of 55 to 65 degrees C. Based on this study, a thermal treatment above 70 degrees C is recommended for crushed vegetable products to prevent oxidation of l-AA to DHAA, the onset of vitamin C degradation. The results reported in this study are applicable for both domestic and industrial processing of vegetables into products such as juices, soups, and purees. In this report, we have demonstrated that processing crushed broccoli in a temperature range of 30 to 60 degrees C could result in the conversion of l-ascorbic acid to dehydroascorbic (DHAA), a very important reaction in regard to vitamin C degradation because DHAA could be easily converted to other compounds that do not have the biological activity of vitamin C.

  10. Variation in the SLC23A1 gene does not influence cardiometabolic outcomes to the extent expected given its association with L-ascorbic acid.

    Science.gov (United States)

    Wade, Kaitlin H; Forouhi, Nita G; Cook, Derek G; Johnson, Paul; McConnachie, Alex; Morris, Richard W; Rodriguez, Santiago; Ye, Zheng; Ebrahim, Shah; Padmanabhan, Sandosh; Watt, Graham; Bruckdorfer, K Richard; Wareham, Nick J; Whincup, Peter H; Chanock, Stephen; Sattar, Naveed; Lawlor, Debbie A; Davey Smith, George; Timpson, Nicholas J

    2015-01-01

    Observational studies showed that circulating L-ascorbic acid (vitamin C) is inversely associated with cardiometabolic traits. However, these studies were susceptible to confounding and reverse causation. We assessed the relation between L-ascorbic acid and 10 cardiometabolic traits by using a single nucleotide polymorphism in the solute carrier family 23 member 1 (SLC23A1) gene (rs33972313) associated with circulating L-ascorbic acid concentrations. The observed association between rs33972313 and cardiometabolic outcomes was compared with that expected given the rs33972313-L-ascorbic acid and L-ascorbic acid-outcome associations. A meta-analysis was performed in the following 5 independent studies: the British Women's Heart and Health Study (n = 1833), the MIDSPAN study (n = 1138), the Ten Towns study (n = 1324), the British Regional Heart Study (n = 2521), and the European Prospective Investigation into Cancer (n = 3737). With the use of a meta-analysis of observational estimates, inverse associations were shown between L-ascorbic acid and systolic blood pressure, triglycerides, and the waist-hip ratio [the strongest of which was the waist-hip ratio (-0.13-SD change; 95% CI: -0.20-, -0.07-SD change; P = 0.0001) per SD increase in L-ascorbic acid], and a positive association was shown with high-density lipoprotein (HDL) cholesterol. The variation at rs33972313 was associated with a 0.18-SD (95% CI: 0.10-, 0.25-SD; P = 3.34 × 10⁻⁶) increase in L-ascorbic acid per effect allele. There was no evidence of a relation between the variation at rs33972313 and any cardiometabolic outcome. Although observed estimates were not statistically different from expected associations between rs33972313 and cardiometabolic outcomes, estimates for low-density lipoprotein cholesterol, HDL cholesterol, triglycerides, glucose, and body mass index were in the opposite direction to those expected. The nature of the genetic association exploited in this study led to limited

  11. Variation in the SLC23A1 gene does not influence cardiometabolic outcomes to the extent expected given its association with l-ascorbic acid1234

    Science.gov (United States)

    Wade, Kaitlin H; Forouhi, Nita G; Cook, Derek G; Johnson, Paul; McConnachie, Alex; Morris, Richard W; Rodriguez, Santiago; Ye, Zheng; Ebrahim, Shah; Padmanabhan, Sandosh; Watt, Graham; Bruckdorfer, K Richard; Wareham, Nick J; Whincup, Peter H; Chanock, Stephen; Sattar, Naveed; Lawlor, Debbie A; Davey Smith, George; Timpson, Nicholas J

    2015-01-01

    Background: Observational studies showed that circulating l-ascorbic acid (vitamin C) is inversely associated with cardiometabolic traits. However, these studies were susceptible to confounding and reverse causation. Objectives: We assessed the relation between l-ascorbic acid and 10 cardiometabolic traits by using a single nucleotide polymorphism in the solute carrier family 23 member 1 (SLC23A1) gene (rs33972313) associated with circulating l-ascorbic acid concentrations. The observed association between rs33972313 and cardiometabolic outcomes was compared with that expected given the rs33972313-l-ascorbic acid and l-ascorbic acid–outcome associations. Design: A meta-analysis was performed in the following 5 independent studies: the British Women's Heart and Health Study (n = 1833), the MIDSPAN study (n = 1138), the Ten Towns study (n = 1324), the British Regional Heart Study (n = 2521), and the European Prospective Investigation into Cancer (n = 3737). Results: With the use of a meta-analysis of observational estimates, inverse associations were shown between l-ascorbic acid and systolic blood pressure, triglycerides, and the waist-hip ratio [the strongest of which was the waist-hip ratio (−0.13-SD change; 95% CI: −0.20-, −0.07-SD change; P = 0.0001) per SD increase in l-ascorbic acid], and a positive association was shown with high-density lipoprotein (HDL) cholesterol. The variation at rs33972313 was associated with a 0.18-SD (95% CI: 0.10-, 0.25-SD; P = 3.34 × 10−6) increase in l-ascorbic acid per effect allele. There was no evidence of a relation between the variation at rs33972313 and any cardiometabolic outcome. Although observed estimates were not statistically different from expected associations between rs33972313 and cardiometabolic outcomes, estimates for low-density lipoprotein cholesterol, HDL cholesterol, triglycerides, glucose, and body mass index were in the opposite direction to those expected. Conclusions: The nature of the genetic

  12. L-ascorbic acid losses in Kenyan vegetables during cooking as determined by high performance liquid chromatography

    OpenAIRE

    N.M.N. Wekesa; S.C. Chhabra; H.M. Thairu

    2001-01-01

    The loss of L-ascorbic acid (L-AA) in 14 different cooked local vegetables found in Nairobi markets was determined by high performance liquid chromatography. The effect of quantity of water on the loss of L-AA during cooking was studied with cowpea leaves. It was found that more L-AA was lost when larger amount of water was used than when smaller amount was used. The effect of the sharpness of the knife on the loss of L-AA was studied with spinach. It was found that more loss of L-AA occurred...

  13. Theoretical study on the adsorption and relative stability of conformers of L-ascorbic acid on γ - alumina (100) surface

    Science.gov (United States)

    Mozaffari Majd, M.; Dabbagh, H. A.; Farrokhpour, H.; Najafi Chermahini, A.

    2017-11-01

    The adsorption energies (Eads) and relative stabilities of selected conformers of the most stable tautomer of L-ascorbic acid (vitamin C) on the dehydroxylated γ-alumina (100) surface were calculated in both gas phase and solvent (water) using the density functional theory (DFT) method. The selected conformers were related to the different rotational angles of OH groups of L-ascorbic acid. The conformational analysis of bare tautomer in both gas and water showed that the conformer No.20 (conf. 20) and 13 (conf. 13) with the dihedral angles of H15sbnd O10sbnd C11sbnd C9 (-73°) and H20sbnd O19sbnd C9sbnd C11 (-135°) were the most stable and unstable conformers, respectively. The performed calculations in the presence of surface showed that the interaction of the conformers with the surface changes their relative stabilities and structures in both gas phase and water. The Ead of each conformer was calculated and it was determined that conf. 8 and conf. 16 have the highest value of Ead in the gas phase (-62.56 kcal/mol) and water (-54.44 kcal/mol), respectively. The optimized structure of each conformer on the surface and the number of hydrogen bonds between it and surface along with their bond lengths were determined.

  14. Molecularly imprinted polyaniline-ferrocene-sulfonic acid-Carbon dots modified pencil graphite electrodes for chiral selective sensing of D-Ascorbic acid and L-Ascorbic acid: A clinical biomarker for preeclampsia

    International Nuclear Information System (INIS)

    Pandey, Indu; Jha, Shashank Shekhar

    2015-01-01

    Highlights: • Pencil graphite electrode was non-covalently functionalized by C-dots. • Electrochemically synthesized ferrocene-sulfonic acid doped PANI film was used as chiral recognition element. • Electrochemical chiral sensing of L-ascorbic acid and D-ascorbic acid was carried out. • L-ascorbic acid determination was done in aqueous, biological and pharmaceutical samples at nM level. - Abstract: A simple and novel method is proposed for chiral separation of L-ascorbic acid and D-ascorbic acid in human cerebrospinal fluids and blood plasma samples. Electro-polymerized molecularly imprinted poly-aniline ferrocenesulfonic acid-C-dots modified pencil graphite electrodes was successfully applied for separation and quantification of D-/L-ascorbic acid in aqueous and some biological samples. Parameters, important to control the performance of the electrochemical sensor were investigated and optimized, including the effects of pH, monomer- template ratios, electropolymerization cycles and scan rates. The molecularly imprinted film exhibited a high chiral selectivity and sensitivity towards D-ascorbic acid and L-ascorbic acid respectively. The surface morphologies and electrochemical properties of the proposed sensor were characterized by scanning electron microscopy, cyclic voltammetry, difference pulse voltammetry, chrono-amperometry and electrochemical impedance spectroscopy. L-ascorbic acid selective sensor shows excellent selectivity towards the L-ascorbic acid in comparison to D- ascorbic acid vice versa for D- ascorbic acid selective sensor. Under optimal conditions the linear range of the calibration curve for L- ascorbic acid and D- ascorbic acid was 6.0–165.0 nM and 6.0–155.0 nM, with the detection limit of 0.001 nM and 0.002 nM. Chiral detection of L-ascorbic acid was successfully carried out in pharmaceuticals and human plasma samples (pregnant women and non pregnant women) via proposed sensor with good selectivity and sensitivity.

  15. Synthesis and characterization of cycloaliphatic hydrophilic polyurethanes, modified with L-ascorbic acid, as materials for soft tissue regeneration

    International Nuclear Information System (INIS)

    Kucinska-Lipka, J.; Gubanska, I.; Strankowski, M.; Cieśliński, H.; Filipowicz, N.; Janik, H.

    2017-01-01

    In this paper we described synthesis and characteristic of obtained hydrophilic polyurethanes (PURs) modified with ascorbic acid (commonly known as vitamin C). Such materials may find an application in the biomedical field, for example in the regenerative medicine of soft tissues, according to ascorbic acid wide influence on tissue regeneration Flora (2009), Szymańska-Pasternak et al. (2011), Taikarimi and Ibrahim (2011), Myrvik and Volk (1954), Li et al. (2001), Cursino et al. (2005) . Hydrophilic PURs were obtained with the use of amorphous α,ω-dihydroxy(ethylene-butylene adipate) (dHEBA) polyol, 1,4-butanediol (BDO) chain extender and aliphatic 4,4′-methylenebis(cyclohexyl isocyanate) (HMDI). HMDI was chosen as a nontoxic diisocyanate, suitable for biomedical PUR synthesis. Modification with L-ascorbic acid (AA) was performed to improve obtained PUR materials biocompatibility. Chemical structure of obtained PURs was provided and confirmed by Fourier transform infrared spectroscopy (FTIR) and Proton nuclear magnetic resonance spectroscopy ( 1 HNMR). Differential scanning calorimetry (DSC) was used to indicate the influence of ascorbic acid modification on such parameters as glass transition temperature, melting temperature and melting enthalpies of obtained materials. To determine how these materials may potentially behave, after implementation in tissue, degradation behavior of obtained PURs in various chemical environments, which were represented by canola oil, saline solution, distilled water and phosphate buffered saline (PBS) was estimated. The influence of AA on hydrophilic-hydrophobic character of obtained PURs was established by contact angle study. This experiment revealed that ascorbic acid significantly improves hydrophilicity of obtained PUR materials and the same cause that they are more suitable candidates for biomedical applications. Good hemocompatibility characteristic of studied PUR materials was confirmed by the hemocompatibility test with

  16. Synthesis and characterization of cycloaliphatic hydrophilic polyurethanes, modified with L-ascorbic acid, as materials for soft tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Kucinska-Lipka, J., E-mail: juskucin@pg.gda.pl [Gdank University of Technology, Faculty of Chemistry, Department of Polymer Technology, Narutowicza St. 11/12, 80-233 Gdansk (Poland); Gubanska, I.; Strankowski, M. [Gdank University of Technology, Faculty of Chemistry, Department of Polymer Technology, Narutowicza St. 11/12, 80-233 Gdansk (Poland); Cieśliński, H.; Filipowicz, N. [Gdansk University of Technology, Faculty of Chemistry, Department of Microbiology, Narutowicza St. 11/12, 80-233 Gdansk (Poland); Janik, H. [Gdank University of Technology, Faculty of Chemistry, Department of Polymer Technology, Narutowicza St. 11/12, 80-233 Gdansk (Poland)

    2017-06-01

    In this paper we described synthesis and characteristic of obtained hydrophilic polyurethanes (PURs) modified with ascorbic acid (commonly known as vitamin C). Such materials may find an application in the biomedical field, for example in the regenerative medicine of soft tissues, according to ascorbic acid wide influence on tissue regeneration Flora (2009), Szymańska-Pasternak et al. (2011), Taikarimi and Ibrahim (2011), Myrvik and Volk (1954), Li et al. (2001), Cursino et al. (2005) . Hydrophilic PURs were obtained with the use of amorphous α,ω-dihydroxy(ethylene-butylene adipate) (dHEBA) polyol, 1,4-butanediol (BDO) chain extender and aliphatic 4,4′-methylenebis(cyclohexyl isocyanate) (HMDI). HMDI was chosen as a nontoxic diisocyanate, suitable for biomedical PUR synthesis. Modification with L-ascorbic acid (AA) was performed to improve obtained PUR materials biocompatibility. Chemical structure of obtained PURs was provided and confirmed by Fourier transform infrared spectroscopy (FTIR) and Proton nuclear magnetic resonance spectroscopy ({sup 1}HNMR). Differential scanning calorimetry (DSC) was used to indicate the influence of ascorbic acid modification on such parameters as glass transition temperature, melting temperature and melting enthalpies of obtained materials. To determine how these materials may potentially behave, after implementation in tissue, degradation behavior of obtained PURs in various chemical environments, which were represented by canola oil, saline solution, distilled water and phosphate buffered saline (PBS) was estimated. The influence of AA on hydrophilic-hydrophobic character of obtained PURs was established by contact angle study. This experiment revealed that ascorbic acid significantly improves hydrophilicity of obtained PUR materials and the same cause that they are more suitable candidates for biomedical applications. Good hemocompatibility characteristic of studied PUR materials was confirmed by the hemocompatibility test

  17. Variation in the SLC23A1 gene does not influence cardiometabolic outcomes to the extent expected given its association with l-ascorbic acid 1 2 3 4

    OpenAIRE

    Wade, Kaitlin H; Forouhi, Nita G; Cook, Derek G; Johnson, Paul; McConnachie, Alex; Morris, Richard W; Rodriguez, Santiago; Ye, Zheng; Ebrahim, Shah; Padmanabhan, Sandosh; Watt, Graham; Bruckdorfer, K Richard; Wareham, Nick J; Whincup, Peter H; Chanock, Stephen

    2014-01-01

    Background: Observational studies showed that circulating l-ascorbic acid (vitamin C) is inversely associated with cardiometabolic traits. However, these studies were susceptible to confounding and reverse causation.\\ud \\ud Objectives:We assessed the relation between l-ascorbic acid and 10 cardiometabolic traits by using a single nucleotide polymorphism in the solute carrier family 23 member 1 (SLC23A1) gene (rs33972313) associated with circulating l-ascorbic acid concentrations. The observed...

  18. Properties of L-ascorbic acid in water and binary aqueous mixtures of D-glucose and D-fructose at different temperatures

    Science.gov (United States)

    Sharma, Ravi; Thakur, R. C.; Sani, Balwinder; Kumar, Harsh

    2017-12-01

    Using density and sound velocity partial molar volumes, partial molar adiabatic compressibilities, partial molar expansibilities and structure of L-ascorbic acid have been determined in water and aqueous mixtures of D-glucose and D-fructose at different concentrations and temperatures. Masson's equation was used to analyze the measured data. The obtained parameters have been interpreted in terms of solute-solute and solute-solvent interactions. It is found that the L-ascorbic acid acts as structure breaker in water as well in binary studied mixtures.

  19. L-ascorbic acid losses in Kenyan vegetables during cooking as determined by high performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    N.M.N. Wekesa

    2001-06-01

    Full Text Available The loss of L-ascorbic acid (L-AA in 14 different cooked local vegetables found in Nairobi markets was determined by high performance liquid chromatography. The effect of quantity of water on the loss of L-AA during cooking was studied with cowpea leaves. It was found that more L-AA was lost when larger amount of water was used than when smaller amount was used. The effect of the sharpness of the knife on the loss of L-AA was studied with spinach. It was found that more loss of L-AA occurred when a blunt (edge thickness 0.08 cm knife was used for cutting the vegetables than when a sharp knife (edge thickness 0.04 cm was used during cooking. L-AA was also determined when vegetables were cooked in different size pieces (surface are >1 cm2

  20. Biosynthesis of l-Ascorbic Acid and Conversion of Carbons 1 and 2 of l-Ascorbic Acid to Oxalic Acid Occurs within Individual Calcium Oxalate Crystal Idioblasts1

    Science.gov (United States)

    Kostman, Todd A.; Tarlyn, Nathan M.; Loewus, Frank A.; Franceschi, Vincent R.

    2001-01-01

    l-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various 14C-labeled compounds and examined by micro-autoradiography for incorporation of 14C into calcium oxalate crystals. [14C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-14C]AsA also gave heavy labeling of crystals, whereas [6-14C]AsA gave no labeling. Labeled precursors of AsA (l-[1-14C]galactose; d-[1-14C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog, d-[1-14C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > l-galactose > d-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis via d-mannose and l-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments. PMID:11161021

  1. Degradation of L-Ascorbic Acid in the Amorphous Solid State.

    Science.gov (United States)

    Sanchez, Juan O; Ismail, Yahya; Christina, Belinda; Mauer, Lisa J

    2018-03-01

    Ascorbic acid degradation in amorphous solid dispersions was compared to its degradation in the crystalline state. Physical blends and lyophiles of ascorbic acid and polymers (pectins and polyvinylpyrrolidone [PVP]) were prepared initially at 50:50 (w/w), with further studies using the polymer that best inhibited ascorbic acid crystallization in the lyophiles in 14 vitamin : PVP ratios. Samples were stored in controlled environments (25 to 60 °C, 0% to 23% RH) for 1 mo and analyzed periodically to track the physical appearance, change in moisture content, physical state (powder x-ray diffraction and polarized light microscopy), and vitamin loss (high performance liquid chromatography) over time. The glass transition temperatures of select samples were determined using differential scanning calorimetry, and moisture sorption profiles were generated. Ascorbic acid in the amorphous form, even in the glassy amorphous state, was more labile than in the crystalline form in some formulations at the highest storage temperature. Lyophiles stored at 25 and 40 °C and those in which ascorbic acid had crystallized at 60 °C (≥70% ascorbic acid : PVP) had no significant difference in vitamin loss (P > 0.05) relative to physical blend controls, and the length of storage had little effect. At 60 °C, amorphous ascorbic acid lyophiles (≤60% ascorbic acid : PVP) lost significantly more vitamin (P vitamin loss significantly increased over time. In these lyophiles, vitamin degradation also significantly increased (P vitamins are naturally present or added at low concentrations and production practices may promote amorphization of the vitamin. Vitamin C is one of the most unstable vitamins in foods. This study documents that amorphous ascorbic acid is less stable than crystalline ascorbic acid in some environments (for example, higher temperatures within 1 wk), especially when the vitamin is present at low concentrations in a product. These findings increase the understanding of

  2. Volumetric and viscometric studies of amino acids in L-ascorbic acid aqueous solutions at T = (293.15 to 323.15) K

    International Nuclear Information System (INIS)

    Jiang, Xiaofeng; Zhu, Chunying; Ma, Youguang

    2014-01-01

    Graphical abstract: The densities and viscosities of L-alanine in L-ascorbic acid aqueous solutions at T = 293.15 K. Highlights: • Densities and viscosities of five amino acids in L-ascorbic acid aqueous solutions were measured. • Based on the experimental data, a series of volumetric and viscometric parameters were calculated. • The group additivity analysis has been applied to analyze the V φ 0 and B-coefficients. -- Abstract: Densities and viscosities of glycine, L-alanine, L-valine, L-threonine and L-arginine in aqueous solutions of (0.0, 0.1, 0.2, 0.3 and 0.4) mol · kg −1 L-ascorbic acid have been measured at T = (293.15, 303.15, 313.15 and 323.15) K under atmospheric pressure. The apparent molar volumes (V φ ), limiting partial molar volumes (V φ 0 ), limiting partial molar volumes of transfer (Δ tr V φ 0 ) and limiting partial molar expansibilities (E 2 0 ) were computed by densities. The extended Jones–Dole equation was used to correlate the viscosities in order to obtain viscosity B-coefficients and the free energies of activation per mole of solvent (Δμ 1 0≠ ) and solute (Δμ 2 0≠ ) were also calculated. The contributions of zwitterionic end group (NH 3 + , COO − ), CH 2 group, OH group and CNHNHNH 2 group to V φ 0 and viscosity B-coefficients were obtained through the group additivity analysis

  3. Electrospray ionization mass spectrometric investigations of [alpha]-dicarbonyl compounds--Probing intermediates formed in the course of the nonenzymatic browning reaction of l-ascorbic acid

    Science.gov (United States)

    Schulz, Anke; Trage, Claudia; Schwarz, Helmut; Kroh, Lothar W.

    2007-05-01

    A new method is presented which allows the simultaneous detection of various [alpha]-dicarbonyl compounds generated in the course of the nonenzymatic browning reaction initiated by thermal treatment of l-ascorbic acid, namely: glyoxal, methylglyoxal, diacetyl, 3-deoxy-l-pentosone, and l-threosoneE 3-Deoxy-l-threosone was successfully identified as a new C4-[alpha]-dicarbonyl structure for the first time in the degradation of Vitamin C by application of this non-chromatographic mass spectrometric approach. Moreover, a more detailed elucidation of the mechanistic scenario with respect to the oxidative and nonoxidative pathways is presented by using dehydro-l-ascorbic acid and 2,3-diketo-l-gulonic acid instead of l-ascorbic acid as a starting material. Furthermore, the postulated pathways are corroborated with the aid of 13C-isotopic labeling studies. The investigations were extended to baby food, and the successful detection of [alpha]-dicarbonyl compounds characteristic for Vitamin C degradation proved the matrix tolerance of the introduced method.

  4. Transcriptomic analysis reveals the metabolic mechanism of L-ascorbic acid in Ziziphus jujuba Mill.

    Directory of Open Access Journals (Sweden)

    Chunmei eZhang

    2016-02-01

    Full Text Available Chinese jujube (Ziziphus jujuba Mill. is the most economically important member of the Rhamnaceae family and contains a high concentration of ascorbic acid (AsA. To explore the metabolic mechanism of AsA accumulation, we investigated the abundance of AsA in the fruit development stages, the leaf and flower of Z. jujuba cv Junzao, and the mature fruit of one type of wild jujube (Z. jujuba var. spinosa Hu, Yanchuan sour jujube. And the expression patterns of genes involved in AsA biosynthesis, degradation and recycling were analyzed. The result showed that AsA biosynthesis during early fruit development (the enlargement stage is the main reason for jujube high accumulation. The L-galactose pathway plays a predominant role in the biosynthesis of AsA during jujube fruit development, and the genes GMP1, GME1, GGP, and GaLDH involved in the determination of AsA concentration during fruit development and in different genotypes; the myo-inositol pathway along with the genes GME2 and GMP2 in the L-galactose pathway play a compensatory role in maintaining AsA accumulation during the ripening stage. These findings enhance our understanding of the molecular mechanism in regulating AsA accumulation for jujube.

  5. Application of l-ascorbic acid and its derivatives (sodium ascorbyl phosphate and magnesium ascorbyl phosphate) in topical cosmetic formulations: stability studies

    International Nuclear Information System (INIS)

    Smaoui, S.; Hilima, H.B.

    2013-01-01

    The present study aimed to formulate and subsequently evaluate a topical skin-care cream (o/w emulsion) from l-ascorbic acid and its derivatives (sodium ascorbyl phosphate and magnesium ascorbyl phosphate) at 2% versus its vehicle (Control). Formulations were developed by entrapping it in the oily phase of o/w emulsion and were stored at 8 degree C, 25 degree C and 40 degree C (in incubator) for a period of four weeks to investigate their stability. In the physical analysis, the evaluation parameters consisted of color, smell, phase separation, centrifugation, and liquefaction. Chemical stability of both derivatives was established by HPLC analysis. In the chemical analysis, the formulation with sodium ascorbyl phosphate was more stable than those with magnesium ascorbyl phosphate and l-ascorbic acid. The microbiological stability of the formulations was also evaluated. The findings indicated that the formulations with l-ascorbic acid and its derivatives were efficient against the proliferation of various spoilage microorganisms, including aerobic plate counts as well as Pseudomonas aeruginosa, Staphylococcus aureus, and yeast and mold counts. The results presented in this work showed good stability throughout the experimental period. Newly formulated emulsion proved to exhibit a number of promising properties and attributes that might open new opportunities for the construction of more efficient, safe, and cost-effective skin-care, cosmetic, and pharmaceutical products. (author)

  6. L-Ascorbic acid metabolism during fruit development in an ascorbate-rich fruit crop chestnut rose (Rosa roxburghii Tratt).

    Science.gov (United States)

    Huang, Ming; Xu, Qiang; Deng, Xiu-Xin

    2014-09-01

    Chestnut rose (Rosa roxburghii Tratt) is a fruit crop that contains unusually high levels of l-ascorbic acid (AsA; ∼1300 mg 100g(-1) FW). To explore the mechanisms underlying AsA metabolism, we investigated the distribution and abundance of AsA during fruit development. We also analyzed gene expression patterns, enzyme activities, and content of metabolites related to AsA biosynthesis and recycling. AsA first accumulated during late fruit development and continued to accumulate during ripening, with the highest accumulation rate near fruit maturity. The redox state of AsA in fruit was also enhanced during late fruit development, while leaf and other tissues had much lower levels of AsA and the redox state of AsA was lower. In mature fruit, AsA was mainly distributed in the cytoplasm of the mesocarp. Correlation analysis suggested that the gene expression patterns, enzyme activities, and related metabolite concentrations involved in the l-galactose pathway showed relatively high correlations with the accumulation rate of AsA. The gene expression pattern and activity of dehydroascorbate reductase (DHAR, EC 1.8.5.1) correlated strongly with AsA concentration, possibly indicating the crucial role of DHAR in the accumulation of high levels of AsA in chestnut rose fruit. Over expression of DHAR in Arabidopsis significantly increased the reduced AsA content and redox state. This was more effective than over expression of the l-galactose pathway gene GDP-d-mannose-3,5-epimerase (EC 5.1.3.18). These findings will enhance understanding of the molecular mechanisms regulating accumulation of AsA in chestnut rose. Copyright © 2014 Elsevier GmbH. All rights reserved.

  7. The role of salicylic acid, L-ascorbic acid and oxalic acid in promoting the oxidation of alkenes with H(2)O(2) catalysed by [Mn(IV) (2)(O)(3)(tmtacn)(2)](2+)

    NARCIS (Netherlands)

    de Boer, Johannes W.; Alsters, Paul L.; Meetsma, Auke; Hage, Ronald; Browne, Wesley R.; Feringa, Ben L.

    2008-01-01

    The role played by the additives salicylic acid, L-ascorbic acid and oxalic acid in promoting the catalytic activity of [Mn(IV) (2)(O)(3)(tmtacn)(2)](PF(6))(2) {1(PF(6))(2), where tmtacn = N, N ', N ''-trimethyl-1,4,7-triazacyclononane} in the epoxidation and cis-dihydroxylation of alkenes with

  8. Investigations to explore interactions in (polyhydroxy solute + L-ascorbic acid + H2O) solutions at different temperatures: Calorimetric and viscometric approach

    International Nuclear Information System (INIS)

    Banipal, Parampaul K.; Sharma, Mousmee; Aggarwal, Neha; Banipal, Tarlok S.

    2016-01-01

    Highlights: • The hydrophilic-hydrophilic interactions predominate at low temperatures. • Enthalpy change for polyol is less exothermic than its parent saccharide. • Δ dil C o p,2,m values suggest structural increase in presence of L-ascorbic acid. • Solutes act as kosmotropes in L-ascorbic acid (aq) solutions as indicated by dB/dT. - Abstract: Isothermal titration micro-calorimeter has been used to measure the enthalpy change (q) of polyhydroxy solutes [(+)-D-xylose, xylitol, (+)-D-glucose, 2-deoxy-D-glucose, (+)-methyl-α-D-glucopyranoside, and (+)-maltose monohydrate] in water and in (0.05, 0.15, and 0.25) mol·kg −1 L-ascorbic acid (aq) solutions at (288.15, 298.15, 308.15, and 318.15) K. Limiting enthalpies of dilution (Δ dil H°) of these solutes were calculated from heat evolved/absorbed during calorimetric experiments. Further thermodynamic quantities such as limiting enthalpies of dilution of transfer (Δ tr Δ dil H°), change in heat capacity (Δ dil C o p,2,m ), and pair (h AB ) and triplet (h ABB ) enthalpic interaction coefficients were also calculated and used to explore the nature of interactions of solutes with cosolute (L-ascorbic acid). The Jones-Dole viscosity B-coefficients for (+)-D-xylose, xylitol, (+)-D-galactose, galactitol, (+)-D-glucose, 2-deoxy-D-glucose, (+)-methyl-α-D-glucopyranoside, and (+)-maltose monohydrate in water and in (0.05, 0.15, 0.25, and 0.35) mol·kg −1 L-ascorbic acid (aq) solutions have been determined from viscosity (η) data measured over temperature range (288.15–318.15) K and at pressure, P = 101.3 kPa. The temperature dependence of B-coefficients (dB/dT), and viscosity B-coefficients of transfer (Δ tr B) of solutes from water to cosolute have also been estimated. These parameters have been discussed in terms of structure-making (kosmotropic) or -breaking (chaotropic) behavior of solutes.

  9. EFFECTS OF L-ASCORBIC ACID AND ALPHA-TOCOPHEROL ON BIOCHEMICAL PARAMETERS OF SWIMMING-INDUCED OXIDATIVE STRESS IN SERUM OF GUINEA PIGS.

    Science.gov (United States)

    Bursać-Mitrović, Marija; Milovanović, Dragan R; Mitić, Radoslav; Jovanović, Danijela; Sovrlić, Miroslav; Vasiljević, Perica; Tomović, Jovica; Manojlović, Nedeljko

    2016-01-01

    The purpose of this study is to determine the effect of L-ascorbic acid and alpha-tocopherol as well as combination of these vitamins with or without exposure to physical exercise on intensity of lipid peroxidation, activity of xanthine oxidase, activity of total antioxidative system, concentration of glutathione, and activity of catalase in the serum of guinea pigs. The experimental measurements of intensity of lipid peroxidation, activity of xanthine oxidase, activity of total antioxidative system, concentration of glutathione, and activity of catalase were done in the serum of guinea pigs. The animals were exposed to the test load to achieve exhaustion and the test was terminated when the animal for the third time to sink into the water. The results of this study demonstrated that endurance exercise of guinea pigs induced oxidative stress response in terms of increased lipid peroxidation and activity of xanthine oxidase in the serum of experimental animals. Our study investigated the antioxidant activity of L-ascorbic acid and alpha-tocopherol also measuring three protective markers in the serum: total antioxidant activity, content of glutathione and activity of catalase. The results obtained show that the vitamins influence the concentrations of above mentioned biochemical parameters, which points out their protective effect of swimming-induced oxidative stress. Single or combined administration of L-ascorbic acid and alpha-tocopherol caused significant inhibition of these markers indicating the important antioxidant activity of the vitamins. Results lead to conclude that the combined treatments with vitamins with or without exposure to physical exercise showed the clear synergistic effect..

  10. Effects of heat stimulation and l-ascorbic acid 2-phosphate supplementation on myogenic differentiation of artificial skeletal muscle tissue constructs.

    Science.gov (United States)

    Ikeda, Kazushi; Ito, Akira; Sato, Masanori; Kanno, Shota; Kawabe, Yoshinori; Kamihira, Masamichi

    2017-05-01

    Although skeletal muscle tissue engineering has been extensively studied, the physical forces produced by tissue-engineered skeletal muscles remain to be improved for potential clinical utility. In this study, we examined the effects of mild heat stimulation and supplementation of a l-ascorbic acid derivative, l-ascorbic acid 2-phosphate (AscP), on myoblast differentiation and physical force generation of tissue-engineered skeletal muscles. Compared with control cultures at 37°C, mouse C2C12 myoblast cells cultured at 39°C enhanced myotube diameter (skeletal muscle hypertrophy), whereas mild heat stimulation did not promote myotube formation (differentiation rate). Conversely, AscP supplementation resulted in an increased differentiation rate but did not induce skeletal muscle hypertrophy. Following combined treatment with mild heat stimulation and AscP supplementation, both skeletal muscle hypertrophy and differentiation rate were enhanced. Moreover, the active tension produced by the tissue-engineered skeletal muscles was improved following combined treatment. These findings indicate that tissue culture using mild heat stimulation and AscP supplementation is a promising approach to enhance the function of tissue-engineered skeletal muscles. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  11. Chemical consequences resulting from multi-millicurie preparation of 6-[18F]-fluoro-6-deoxy-L-ascorbic acid

    International Nuclear Information System (INIS)

    Kothari, P.J.; Finn, R.D.; Bornmann, W.G.; Agus, D.B.; Vera, J.C.; Larson, S.M.

    1997-01-01

    Ascorbic acid is required for human physiology and is particularly important in maintaining normal connective tissue and host defense. Our interest in this carboxylic acid stems from its similarity in transport with glucose in human cells. In order to investigate these phenomena, we report a refined synthesis of 6-[ 18 F]-fluoro-6-deoxy-L-ascorbic acid ( 18 F-FAA) via nucleophilic displacement of the cyclic sulfate with Kryptofix 2.2.2/K 18 F complex which results in multi-millicurie quantities of high specific activity product. The trace carrier-added synthesis has resulted in a decay corrected radiochemical yield of 31.6±3.4% with a specific activity approaching 180 mCi/μmol. Our preliminary experience with the assessment of the chemical stability of both the radiolabelled and stable product is also presented. (orig.)

  12. Different response to acetic acid stress in Saccharomyces cerevisiae wild-type and l-ascorbic acid-producing strains.

    Science.gov (United States)

    Martani, Francesca; Fossati, Tiziana; Posteri, Riccardo; Signori, Lorenzo; Porro, Danilo; Branduardi, Paola

    2013-09-01

    Biotechnological processes are of increasing significance for industrial production of fine and bulk chemicals, including biofuels. Unfortunately, under operative conditions microorganisms meet multiple stresses, such as non-optimal pH, temperature, oxygenation and osmotic stress. Moreover, they have to face inhibitory compounds released during the pretreatment of lignocellulosic biomasses, which constitute the preferential substrate for second-generation processes. Inhibitors include furan derivatives, phenolic compounds and weak organic acids, among which acetic acid is one of the most abundant and detrimental for cells. They impair cellular metabolism and growth, reducing the productivity of the process: therefore, the development of robust cell factories with improved production rates and resistance is of crucial importance. Here we show that a yeast strain engineered to endogenously produce vitamin C exhibits an increased tolerance compared to the parental strain when exposed to acetic acid at moderately toxic concentrations, measured as viability on plates. Starting from this evidence, we investigated more deeply: (a) the nature and levels of reactive oxygen species (ROS); (b) the activation of enzymes that act directly as detoxifiers of reactive oxygen species, such as superoxide dismutase (SOD) and catalase, in parental and engineered strains during acetic acid stress. The data indicate that the engineered strain can better recover from stress by limiting ROS accumulation, independently from SOD activation. The engineered yeast can be proposed as a model for further investigating direct and indirect mechanism(s) by which an antioxidant can rescue cells from organic acid damage; moreover, these studies will possibly provide additional targets for further strain improvements. Copyright © 2013 John Wiley & Sons, Ltd.

  13. Electrocatalytic determination of L-ascorbic acid by modified glassy carbon with Ni(Me2(CH3CO)2[14]tetraenoN4) complex.

    Science.gov (United States)

    Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam

    2003-12-01

    A novel modified glassy carbon electrode containing Ni(Me2(CH3CO)2[14]tetraenoN4) complex was used as an electrocatalytic sensor for the determination of L-ascorbic acid in pH = 6.6. The peak potential shifted to negative by 205 mV compared with that for a bare electrode in cyclic voltammograms. The calibration curve was linear up to 6.2 x 10(-3) M with a detection limit 3.1 x 10(-7) M and an RSD% better than 2.47%. This newly modified electrode was applied to commercial pharmaceutical tablets, injections and foods. The obtained results were identical to those obtained by the classical 2,6-dichlorophenolindophenol method.

  14. L-dehydroascorbic acid can substitute l-ascorbic acid as dietary vitamin C source in guinea pigs.

    Science.gov (United States)

    Frikke-Schmidt, Henriette; Tveden-Nyborg, Pernille; Lykkesfeldt, Jens

    2016-04-01

    Vitamin C deficiency globally affects several hundred million people and has been associated with increased morbidity and mortality in numerous studies. In this study, bioavailability of the oxidized form of vitamin C (l-dehydroascorbic acid or DHA)-commonly found in vitamin C containing food products prone to oxidation-was studied. Our aim was to compare tissue accumulation of vitamin C in guinea pigs receiving different oral doses of either ascorbate or DHA. In all tissues tested (plasma, liver, spleen, lung, adrenal glands, kidney, muscle, heart, and brain), only sporadic differences in vitamin C accumulation from ascorbate or DHA were observed except for the lowest dose of DHA (0.25mg/ml in the drinking water), where approximately half of the tissues had slightly yet significantly less vitamin C accumulation than from the ascorbate source. As these results contradicted data from rats, we continued to explore the ability to recycle DHA in blood, liver and intestine in guinea pigs, rats and mice. These investigations revealed that guinea pigs have similar recycling capacity in red blood cells as observed in humans, while rats and mice do not have near the same ability to reduce DHA in erythrocytes. In liver and intestinal homogenates, guinea pigs also showed a significantly higher ability to recycle DHA compared to rats and mice. These data demonstrate that DHA in guinea pigs-as in humans-is almost as effective as ascorbate as vitamin C source when it comes to taking up and storing vitamin C and further suggest that the guinea pig is superior to other rodents in modeling human vitamin C homeostasis. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Effect of quinolinic acid in the nucleus basalis magnocellularis on cortical high-affinity choline uptake

    Energy Technology Data Exchange (ETDEWEB)

    Metcalf, R.H.; Boegman, R.J.; Quirion, R.; Riopelle, R.J.; Ludwin, S.K.

    1987-08-01

    A transient 45% increase in cortical high-affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific (/sup 3/H)hemicholinium-3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.

  16. Syringe needle-based sampling coupled with liquid-phase extraction for determination of the three-dimensional distribution of l-ascorbic acid in apples.

    Science.gov (United States)

    Tang, Sheng; Lee, Hian Kee

    2016-05-15

    A novel syringe needle-based sampling approach coupled with liquid-phase extraction (NBS-LPE) was developed and applied to the extraction of l-ascorbic acid (AsA) in apple. In NBS-LPE, only a small amount of apple flesh (ca. 10mg) was sampled directly using a syringe needle and placed in a glass insert for liquid extraction of AsA by 80 μL oxalic acid-acetic acid. The extract was then directly analyzed by liquid chromatography. This new procedure is simple, convenient, almost organic solvent free, and causes far less damage to the fruit. To demonstrate the applicability of NBS-LPE, AsA levels at different sampling points in a single apple were determined to reveal the spatial distribution of the analyte in a three-dimensional model. The results also showed that this method had good sensitivity (limit of detection of 0.0097 mg/100g; limit of quantification of 0.0323 mg/100g), acceptable reproducibility (relative standard deviation of 5.01% (n=6)), a wide linear range of between 0.05 and 50mg/100g, and good linearity (r(2)=0.9921). This interesting extraction technique and modeling approach can be used to measure and monitor a wide range of compounds in various parts of different soft-matrix fruits and vegetables, including single specimens. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Light exposure during storage preserving soluble sugar and l-ascorbic acid content of minimally processed romaine lettuce (Lactuca sativa L.var. longifolia).

    Science.gov (United States)

    Zhan, Lijuan; Hu, Jinqiang; Ai, Zhilu; Pang, Lingyun; Li, Yu; Zhu, Meiyun

    2013-01-01

    Minimally processed romaine lettuce (MPRL) leaves were stored in light condition (2500lux) or darkness at 4°C for 7d. Light exposure significantly delayed the degradation of chlorophyll and decrease of glucose, reducing sugar, and sucrose content, and thus preserved more total soluble solid (TSS) content at the end of storage in comparison with darkness. While, it did not influenced starch content that progressively decreased over time. The l-ascorbic acid (AA) accumulated in light-stored leaves, but deteriorated in dark-stored leaves during storage. The dehydroascorbic acid (DHA) increased in all leaves stored in both light and dark condition, of which light condition resulted in less DHA than darkness. In addition, the fresh weight loss and dry matter significantly increased and these increases were accelerated by light exposure. Conclusively, light exposure in applied intensity effectively alleviated MPRL quality deterioration by delaying the decreases of pigments, soluble sugar, TSS content and accumulating AA. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Sawake, Shota [Saitama Univ. (Japan); Tajima, Noriaki [Saitama Univ. (Japan); Mortimer, Jenny C. [Univ. of Cambridge (United Kingdom); RIKEN Center for Sustainable Resource Science, Yokohama (Japan); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lao, Jeemeng [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Ishikawa, Toshiki [Saitama Univ. (Japan); Yu, Xiaolan [Univ. of Cambridge (United Kingdom); Yamanashi, Yukiko [Saitama Univ. (Japan); Yoshimi, Yoshihisa [Saitama Univ. (Japan); Kawai-Yamada, Maki [Saitama Univ. (Japan); Dupree, Paul [Saitama Univ. (Japan); Tsumuraya, Yoichi [Saitama Univ. (Japan); Kotake, Toshihisa [Saitama Univ. (Japan); Univ. of Cambridge (United Kingdom)

    2015-12-01

    Humans are unable to synthesize L-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.

  19. Effect of Dietary L-ascorbic Acid (L-AA on Production Performance, Egg Quality Traits and Fertility in Japanese Quail ( at Low Ambient Temperature

    Directory of Open Access Journals (Sweden)

    N. Shit

    2012-07-01

    Full Text Available Environmental stress boosts the levels of stress hormones and accelerates energy expenditure which subsequently imbalance the body’s homeostasis. L-ascorbic acid (L-AA has been recognized to mitigate the negative impact of environmental stress on production performances in birds. The present investigation was carried out to elucidate the effect of different dietary levels of L-AA on production performance, egg quality traits and fertility in Japanese quail at low ambient temperature. Sixty matured females (15 wks were equally divided into three groups (20/group based on the different dietary levels of L-AA (0, 250 and 500 ppm and coupled with an equal number of males (1:1 obtained from the same hatch. They were managed in uniform husbandry conditions without restriction of feed and water at 14 h photo-schedule. Except for feed efficiency, body weight change, feed consumption and hen-day egg production were recorded highest in 500 ppm L-AA supplemented groups. Among the all egg quality traits studied, only specific gravity, shell weight and thickness differed significantly (p<0.05 in the present study. Fertility was improved significantly (p<0.01 to a dose dependent manner of L-AA. The findings of the present study concluded that dietary L-AA can be a caring management practice at least in part to alleviate the adverse effect of cold induced stress on production performance in Japanese quail.

  20. Positron-labeled antioxidant 6-deoxy-6-[{sup 18}F]fluoro-L-ascorbic acid: Increased uptake in transient global ischemic rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Fumihiko; Shibata, Shigenobu; Watanabe, Shigenori; Masuda, Kouji; Maeda, Minoru

    1996-05-01

    The in vivo uptake and distribution of 6-deoxy-6-[{sup 18}F]fluoro-L-ascorbic acid ({sup 18}F-DFA) were investigated in rat brains following postischemic reperfusion. Global cerebral ischemia was induced in male Wistar rats for 20 min by occlusion of four major arteries. Two time points were chosen for {sup 18}F-DFA injection to rats subjected to cerebral ischemia, at the start of recirculation and 5 days following recirculation. The rats were then killed at 2 h after tail-vein administration of {sup 18}F-DFA and tissue radioactivity concentration was determined. Increased uptake of radioactivity in particular brain regions, including the cerebral cortex, hypothalamus, and amygdala following injection of {sup 18}F-DFA, compared to the sham-operated control, was observed 5 days after reperfusion. Similar results were also obtained in in vitro experiments using brain slices. Abnormal in vivo accumulation of {sup 45}Ca, a marker of regional postischemic injury, was observed in these brain regions in tissue dissection experiments. Furthermore, metabolite analysis of nonradioactive DFA using {sup 19}F-NMR showed that DFA remained intact in the postischemic reperfusion brain. The present results indicate that {sup 18}F-DFA increasingly accumulates in damaged regions of postischemic reperfusion brain.

  1. 2-O-α-glucopytanosyl L-ascorbic acid reduced mutagenicity at HPRT locus of mouse splenocytes following BNCT

    International Nuclear Information System (INIS)

    Kinashi, Yuko; Masunaga, Shin-ichiro; Suzuki, Minoru; Nagata, Kanji; Ono, Koji

    2006-01-01

    In boron neutron capture therapy (BNCT), normal tissue surrounding the tumor cells sometimes take up boron compounds resulting in radiation-induced damage to normal tissue. We have previously reported the evidence for increased the mutagenicity of thermal neutron in the presence of boron. In addition, we described the biological radio-protective effects of the ascorbic acid for mutation induction following BNCT in vitro. Here, we investigated these radio-protective effects of ascorbic acid for mutation induction in mouse splenocytes on HPRT locus following a BNCT study in vivo. (author)

  2. KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis.

    Science.gov (United States)

    Sawake, Shota; Tajima, Noriaki; Mortimer, Jenny C; Lao, Jeemeng; Ishikawa, Toshiki; Yu, Xiaolan; Yamanashi, Yukiko; Yoshimi, Yoshihisa; Kawai-Yamada, Maki; Dupree, Paul; Tsumuraya, Yoichi; Kotake, Toshihisa

    2015-12-01

    Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity. © 2015 American Society of Plant Biologists. All rights reserved.

  3. Theoretical study of the changes in the vibrational characteristics arising from the hydrogen bonding between Vitamin C ( L-ascorbic acid) and H 2O

    Science.gov (United States)

    Dimitrova, Yordanka

    2006-02-01

    The vibrational characteristics (vibrational frequencies, infrared intensities and Raman activities) for the hydrogen-bonded system of Vitamin C ( L-ascorbic acid) with five water molecules have been predicted using ab initio SCF/6-31G(d, p) calculations and DFT (BLYP) calculations with 6-31G(d, p) and 6-31++G(d, p) basis sets. The changes in the vibrational characteristics from free monomers to a complex have been calculated. The ab initio and BLYP calculations show that the complexation between Vitamin C and five water molecules leads to large red shifts of the stretching vibrations for the monomer bonds involved in the hydrogen bonding and very strong increase in their IR intensity. The predicted frequency shifts for the stretching vibrations from Vitamin C taking part in the hydrogen bonding are up to -508 cm -1. The magnitude of the wavenumber shifts is indicative of relatively strong OH···H hydrogen-bonded interactions. In the same time the IR intensity and Raman activity of these vibrations increase upon complexation. The IR intensity increases dramatically (up to 12 times) and Raman activity increases up to three times. The ab initio and BLYP calculations show, that the symmetric OH vibrations of water molecules are more sensitive to the complexation. The hydrogen bonding leads to very large red shifts of these vibrations and very strong increase in their IR intensity. The asymmetric OH stretching vibrations of water, free from hydrogen bonding are less sensitive to the complexation than the hydrogen-bonded symmetric O sbnd H stretching vibrations. The increases of the IR intensities for these vibrations are lower and red shifts are negligible.

  4. Final report of the safety assessment of L-Ascorbic Acid, Calcium Ascorbate, Magnesium Ascorbate, Magnesium Ascorbyl Phosphate, Sodium Ascorbate, and Sodium Ascorbyl Phosphate as used in cosmetics.

    Science.gov (United States)

    Elmore, Amy R

    2005-01-01

    L-Ascorbic Acid, Calcium Ascorbate, Magnesium Ascorbate, Magnesium Ascorbyl Phosphate, Sodium Ascorbate, and Sodium Ascorbyl Phosphate function in cosmetic formulations primarily as antioxidants. Ascorbic Acid is commonly called Vitamin C. Ascorbic Acid is used as an antioxidant and pH adjuster in a large variety of cosmetic formulations, over 3/4 of which were hair dyes and colors at concentrations between 0.3% and 0.6%. For other uses, the reported concentrations were either very low (cosmetics, but are not currently used. Sodium Ascorbyl Phosphate functions as an antioxidant in cosmetic products and is used at concentrations ranging from 0.01% to 3%. Magnesium Ascorbyl Phosphate functions as an antioxidant in cosmetics and was reported being used at concentrations from 0.001% to 3%. Sodium Ascorbate also functions as an antioxidant in cosmetics at concentrations from 0.0003% to 0.3%. Related ingredients (Ascorbyl Palmitate, Ascorbyl Dipalmitate, Ascorbyl Stearate, Erythorbic Acid, and Sodium Erythorbate) have been previously reviewed by the Cosmetic Ingredient Review (CIR) Expert Panel and found "to be safe for use as cosmetic ingredients in the present practices of good use." Ascorbic Acid is a generally recognized as safe (GRAS) substance for use as a chemical preservative in foods and as a nutrient and/or dietary supplement. Calcium Ascorbate and Sodium Ascorbate are listed as GRAS substances for use as chemical preservatives. L-Ascorbic Acid is readily and reversibly oxidized to L-dehydroascorbic acid and both forms exist in equilibrium in the body. Permeation rates of Ascorbic Acid through whole and stripped mouse skin were 3.43 +/- 0.74 microg/cm(2)/h and 33.2 +/- 5.2 microg/cm(2)/h. Acute oral and parenteral studies in mice, rats, rabbits, guinea pigs, dogs, and cats demonstrated little toxicity. Ascorbic Acid and Sodium Ascorbate acted as a nitrosation inhibitor in several food and cosmetic product studies. No compound-related clinical signs or gross or

  5. Humic Acid Confers HIGH-AFFINITY K+ TRANSPORTER 1-Mediated Salinity Stress Tolerance in Arabidopsis.

    Science.gov (United States)

    Khaleda, Laila; Park, Hee Jin; Yun, Dae-Jin; Jeon, Jong-Rok; Kim, Min Gab; Cha, Joon-Yung; Kim, Woe-Yeon

    2017-12-31

    Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on Arabidopsis thaliana seedlings. To identify the molecular mechanisms of HA-induced salt stress tolerance in Arabidopsis, we examined possible roles of a sodium influx transporter HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1). Salt-induced root growth inhibition in HKT1 overexpressor transgenic plants (HKT1-OX) was rescued by application of HA, but not in wild-type and other plants. Moreover, salt-induced degradation of HKT1 protein was blocked by HA treatment. In addition, the application of HA to HKT1-OX seedlings led to increased distribution of Na+ in roots up to the elongation zone and caused the reabsorption of Na+ by xylem and parenchyma cells. Both the influx of the secondary messenger calcium and its cytosolic release appear to function in the destabilization of HKT1 protein under salt stress. Taken together, these results suggest that HA could be applied to the field to enhance plant growth and salt stress tolerance via post-transcriptional control of the HKT1 transporter gene under saline conditions.

  6. 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...

  7. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. De novo assembly and functional annotation of Myrciaria dubia fruit transcriptome reveals multiple metabolic pathways for L-ascorbic acid biosynthesis.

    Science.gov (United States)

    Castro, Juan C; Maddox, J Dylan; Cobos, Marianela; Requena, David; Zimic, Mirko; Bombarely, Aureliano; Imán, Sixto A; Cerdeira, Luis A; Medina, Andersson E

    2015-11-24

    Myrciaria dubia is an Amazonian fruit shrub that produces numerous bioactive phytochemicals, but is best known by its high L-ascorbic acid (AsA) content in fruits. Pronounced variation in AsA content has been observed both within and among individuals, but the genetic factors responsible for this variation are largely unknown. The goals of this research, therefore, were to assemble, characterize, and annotate the fruit transcriptome of M. dubia in order to reconstruct metabolic pathways and determine if multiple pathways contribute to AsA biosynthesis. In total 24,551,882 high-quality sequence reads were de novo assembled into 70,048 unigenes (mean length = 1150 bp, N50 = 1775 bp). Assembled sequences were annotated using BLASTX against public databases such as TAIR, GR-protein, FB, MGI, RGD, ZFIN, SGN, WB, TIGR_CMR, and JCVI-CMR with 75.2 % of unigenes having annotations. Of the three core GO annotation categories, biological processes comprised 53.6 % of the total assigned annotations, whereas cellular components and molecular functions comprised 23.3 and 23.1 %, respectively. Based on the KEGG pathway assignment of the functionally annotated transcripts, five metabolic pathways for AsA biosynthesis were identified: animal-like pathway, myo-inositol pathway, L-gulose pathway, D-mannose/L-galactose pathway, and uronic acid pathway. All transcripts coding enzymes involved in the ascorbate-glutathione cycle were also identified. Finally, we used the assembly to identified 6314 genic microsatellites and 23,481 high quality SNPs. This study describes the first next-generation sequencing effort and transcriptome annotation of a non-model Amazonian plant that is relevant for AsA production and other bioactive phytochemicals. Genes encoding key enzymes were successfully identified and metabolic pathways involved in biosynthesis of AsA, anthocyanins, and other metabolic pathways have been reconstructed. The identification of these genes and pathways is in agreement with

  9. Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G).

    Science.gov (United States)

    Han, Ruizhi; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Chen, Jian

    2013-07-01

    In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of L-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2% higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at -3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.

  10. 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.

  11. Locked nucleic acid (LNA): High affinity targeting of RNA for diagnostics and therapeutics

    DEFF Research Database (Denmark)

    Kauppinen, S.; Vester, Birte; Wengel, Jesper

    2005-01-01

    Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. This conformational restriction results in unprecedented hybridization affinity towards complementary single stranded RN...

  12. BAHAN PENYERAP KMnO4 DAN ASAM L-ASKORBAT DALAM PENGEMASAN AKTIF (ACTIVE PACKAGING UNTUK MEMPERPANJANG MASA SIMPAN DAN MEMPERTAHANKAN MUTU BUAH DUKU (Lansium domesticum Corr. [Adsorbers for KMnO4 and L-Ascorbic Acid in the Active Packaging to Prolong the Shelve-Life and Maintain the Quality of Lanzone (Lansium domesticum Corr. Fruits

    Directory of Open Access Journals (Sweden)

    Soesiladi E Widodo

    2005-08-01

    Full Text Available To develop an active packaging of lanzone (Lansium domisticum Corr. Fruits, KmnO4 as an ethylene scavenger and L-ascorbic acid as an oxygent scavenger were inserted into packaging. As direct contact of KmnO4 with agricultural product was not recommended and due to the liquid characteristic of both scavenger was carried out. This research was aimed at finding out the best adsorbers for KmnO4, L-ascorbic acid, and their combination in an active packaging to prolog the shelve-life and to maintain the quality of lanzone fruits. The result showed that 1 among the four adsorbers tested, pumice could was the best alternative as a KmnO4 or L-ascorbic acid adsorbers, and 2 spon and pumice were the best alternative adsorber for the combination of KmnO4 or L-ascorbic acid. Both adsorber were effective in prolonging the shelve-live (8-11 days longer than with out packaging and as good as using silica gel and vermiculite and maintaining the quality of lanzone fruits.

  13. Ácido L-ascórbico: reações de complexação e de óxido-redução com alguns íons metálicos de transição L-ascorbic acid: complexation and redox reactions with some transition metal ions

    OpenAIRE

    Adalgiza Fornaro; Nina Coichev

    1998-01-01

    The strong reducing action of L-ascorbic acid (Vitamin C) are of fundamental interest in biochemical and related process. The oxidation of ascorbic acid by molecular oxygen and others oxidants are of fundamental importance, involving the intervention of transition metal ions as catalysts and the formation transition metal complexes of ascorbic acid as intermediates. The present article is intended to cover some aspects of the reactions of ascorbic acid and related compounds involving some tra...

  14. Antiaging effects of a novel facial serum containing L-ascorbic acid, proteoglycans, and proteoglycan-stimulating tripeptide: ex vivo skin explant studies and in vivo clinical studies in women

    Directory of Open Access Journals (Sweden)

    Garre A

    2018-05-01

    Full Text Available Aurora Garre,1 Mridvika Narda,1 Palmira Valderas-Martinez,1 Jaime Piquero,2 Corinne Granger1 1Innovation and Development, ISDIN SA, Barcelona, Spain; 2Dermik Clinic, Barcelona, Spain Background: With age, decreasing dermal levels of proteoglycans, collagen, and elastin lead to the appearance of aged skin. Oxidation, largely driven by environmental factors, plays a central role.Aim: The aim of this study was to assess the antiaging efficacy of a topical serum containing l-ascorbic acid, soluble proteoglycans, low molecular weight hyaluronic acid, and a tripeptide in ex vivo and in vivo clinical studies.Methods: Photoaging and photo-oxidative damage were induced in human skin explants by artificial solar radiation. Markers of oxidative stress – reactive oxygen species (ROS, total glutathione (GSH, and cyclobutane pyrimidine dimers (CPDs – were measured in serum-treated explants and untreated controls. Chronological aging was simulated using hydrocortisone. In both ex vivo studies, collagen, elastin, and proteoglycans were determined as measures of dermal matrix degradation. In women aged 21–67 years, hydration was measured up to 24 hours after a single application of serum, using Corneometer and hygrometer. Subjects’ perceptions of efficacy and acceptability were assessed via questionnaire after once-daily serum application for 4 weeks. Studies were performed under the supervision of a dermatologist.Results: In the photoaging study, irradiation induced changes in ROS, CPD, GSH, collagen, and elastin levels; these changes were reversed by topical serum application. The serum also protected against hydrocortisone-induced reduction in collagen, elastin, and proteoglycan levels, which were significantly higher in the serum-treated group vs untreated hydrocortisone-control explants. In clinical studies, serum application significantly increased skin moisture for 6 hours. Healthy volunteers perceived the product as efficient in making the

  15. High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

    Science.gov (United States)

    Goldman, Johnathan M; Zhang, Li Ang; Manna, Arunava; Armitage, Bruce A; Ly, Danith H; Schneider, James W

    2013-07-08

    Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

  16. Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand

    NARCIS (Netherlands)

    Dekker, Frank J; de Mol, Nico J; Fischer, Marcel J E; Liskamp, Rob M J; Dekker, Frank

    2003-01-01

    The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The

  17. Measurement and Correlation on Viscosity and Apparent Molar Volume of Ternary System for L-ascorbic Acid in Aqueous D-Glucose and Sucrose Solutions%L-抗坏血酸在葡萄糖和蔗糖溶液中的黏度及其热力学性质的研究

    Institute of Scientific and Technical Information of China (English)

    赵长伟; 马沛生

    2003-01-01

    Viscosities and densities at several temperatures from 293.15 K to 313.15 K are reported for L-ascorbic acid in aqueous glucose and sucrose solutions at different concentrations. The parameters of density, viscosity coefficient B and partial molar volume are calculated by regression. The experimental results show that densities and viscosities decrease as temperature increases at the same solute and solvent (glucose and sucrose aqueous solution) concentrations, and increase with concentration of glucose and sucrose at the same solute concentration and temperature. B increases with concentration of glucose and sucrose and temperature. L-ascorbic acid is structure-breaker or structure-making for the glucose and sucrose aqueous solutions. Furthermore, the solute-solvent interactions in ternary systems of water-glucose-electrolyte and water-sucrose-electrolyte are discussed.

  18. Effects of combined treatment of α-tocopherol, L-ascorbic acid, selenium and zinc on bleomycin, etoposide and cisplatin-induced alterations in testosterone synthesis pathway in rats.

    Science.gov (United States)

    Kilarkaje, Narayana

    2014-12-01

    To investigate the effects of therapeutically relevant dose levels of bleomycin, etoposide and cisplatin (BEP) on testicular steroidogenic enzymes, and possible protective effects of an antioxidant cocktail (AC). Adult Sprague-Dawley rats received BEP with or without the AC (α-tocopherol, L-ascorbic acid, selenium and zinc) for either (a) 4 days (short term; 1.5, 15 and 3 mg/kg), or (b) three cycles of 21 days each (0.75, 7.5 and 1.5 mg/kg), or (c) the three cycles with a 63-day recovery period. The expression of steroidogenic enzymes were measured in the testes by Western blotting and immunofluorescent labeling. The short-term BEP exposure resulted in a decrease in scavenger receptor class-B1 and an increase in luteinizing hormone receptor (LHR). The AC with or without BEP has increased the levels of LHR, 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-HSD, but without significant changes in testosterone levels. The three cycles of BEP up-regulated the expression of steroidogenic acute regulatory protein (StAR) and down-regulated that of cholesterol side chain cleavage enzyme (P450scc), cytochrome p450 17A1 (Cyp17A1, recovered by the AC) and 17β-HSD, associated with significant reduction in testosterone levels. The three cycles with the recovery time led to decreases in LHR, StAR, P450scc and Cyp17A1 and increases in 3β-HSD and 17β-HSD. The AC did not enhance the recovery of the enzyme levels. The three cycles of BEP treatment inhibit the testosterone synthesis pathway even after the recovery time. The AC recovers the effects of BEP chemotherapy on a few steroidogenic enzymes.

  19. Thermal expansion of L-ascorbic acid

    Science.gov (United States)

    Nicolaï, B.; Barrio, M.; Tamarit, J.-Ll.; Céolin, R.; Rietveld, I. B.

    2017-04-01

    The specific volume of vitamin C has been investigated by X-ray powder diffraction as a function of temperature from 110 K up to complete degradation around 440 K. Its thermal expansion is relatively small in comparison with other organic compounds with an expansivity α v of 1.2(3) × 10-4 K-1. The structure consists of strongly bound molecules in the ac plane through a dense network of hydrogen bonds. The thermal expansion is anisotropic. Along the b axis, the expansion has most leeway and is about 10 times larger than in the other directions.

  20. Na+-Dependent High-Affinity Nitrate, Phosphate and Amino Acids Transport in Leaf Cells of the Seagrass Posidonia oceanica (L. Delile

    Directory of Open Access Journals (Sweden)

    Lourdes Rubio

    2018-05-01

    Full Text Available Posidonia oceanica (L. Delile is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate and phosphate. Classical depletion experiments, membrane potential and cytosolic sodium measurements were used to characterize the high-affinity NO3−, Pi and amino acids uptake mechanisms in this species. Net uptake rates of both NO3− and Pi were reduced by more than 70% in the absence of Na+. Micromolar concentrations of NO3− depolarized mesophyll leaf cells plasma membrane. Depolarizations showed saturation kinetics (Km = 8.7 ± 1 μM NO3−, which were not observed in the absence of Na+. NO3− induced depolarizations at increasing Na+ also showed saturation kinetics (Km = 7.2 ± 2 mM Na+. Cytosolic Na+ measured in P. oceanica leaf cells (17 ± 2 mM Na+ increased by 0.4 ± 0.2 mM Na+ upon the addition of 100 μM NO3−. Na+-dependence was also observed for high-affinity l-ala and l-cys uptake and high-affinity Pi transport. All together, these results strongly suggest that NO3−, amino acids and Pi uptake in P. oceanica leaf cells are mediated by high-affinity Na+-dependent transport systems. This mechanism seems to be a key step in the process of adaptation of seagrasses to the marine environment.

  1. 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 ...

  2. L-ascorbic acid, β-carotene and lycopene content in papaya fruits (Carica papaya with or without physiological skin freckles Conteúdo de ácido l-ascórbico, β-caroteno e licopeno em frutos de mamão (Carica papaya com e sem mancha fisiológica

    Directory of Open Access Journals (Sweden)

    Leandro Marelli de Souza

    2008-01-01

    Full Text Available The Skin Freckles is a papaya skin disorder that depreciates de fruit appearance and hampers its commercialization, although not lowering its nutritive value. Being the papaya a good source of ascorbic acid, β-carotene and licopene this research aimed at determining L-ascorbic acid, β-carotene and licopene content in papaya fruits, from 'Formosa' and 'Solo' group varieties, with and without apparent physiological skin disease (skin freckles. Fruits were harvested in the Southeast Region of Brazil. L-ascorbic acid content was determined by titration technique. β-carotene and licopene contents were determined by high performance liquid chromatography technique (HPLC. L-ascorbic acid content in papaya fruits ranged from (59.9 ± 3.4 mg 100 g-1 to (112.4 ± 12.6 mg 100 g-1 in fresh papaya pulp. β-carotene content ranged from (0.19 ± 0.07 mg 100 g-1 to (0.56 ± 0.09 mg 100 g-1 and that of licopene ranged from (1.44 ± 0.28 mg 100 g-1 to (3.39 ± 0.32 mg 100 g-1 in fresh papaya pulp. L-ascorbic acid contents of papaya fruits with skin disease averaged 7.0 mg 100 g-1 to 10.0 mg 100 g-1 higher than those of papaya fruits without skin freckles (P A Mancha Fisiológica do Mamão (MFM é uma desordem da casca do mamão, que deprecia a aparência do fruto e prejudica a sua comercialização, embora não prejudique o seu valor nutritivo. Considerando ser o mamão, uma boa fonte de ácido L-ascórbico, b-caroteno e licopeno, esta pesquisa visou determinar o índice destes componentes em frutas de mamão, das variedades do grupo 'Formosa' e 'Solo', com e sem Mancha Fisiológica do Mamão (MFM aparente na pele. As frutas foram colhidas na região do sudeste de Brasil. O teor de ácido L-ascórbico foi determinado pela técnica de titulação. Os índices do b-caroteno e do licopeno foram determinados pela técnica de cromatografia líquida de alta eficiência (HPLC. O teor de ácido L-ascórbico variou de (59,9 ± 3,4 mg 100 g-1 a (112,4 ± 12,6 mg 100 g-1

  3. Identification and functional analysis of the L-ascorbate-specific enzyme II complex of the phosphotransferase system in Streptococcus mutans.

    Science.gov (United States)

    Wu, Xinyu; Hou, Jin; Chen, Xiaodan; Chen, Xuan; Zhao, Wanghong

    2016-03-22

    Streptococcus mutans is the primary etiological agent of human dental caries. It can metabolize a wide variety of carbohydrates and produce large amounts of organic acids that cause enamel demineralization. Phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays an important role in carbohydrates uptake of S. mutans. The ptxA and ptxB genes in S. mutans encode putative enzyme IIA and enzyme IIB of the L-ascorbate-specific PTS. The aim of this study was to analyze the function of these proteins and understand the transcriptional regulatory mechanism. ptxA (-), ptxB (-), as well as ptxA (-) , ptxB (-) double-deletion mutants all had more extended lag phase and lower growth yield than wild-type strain UA159 when grown in the medium using L-ascorbate as the sole carbon source. Acid production and acid killing assays showed that the absence of the ptxA and ptxB genes resulted in a reduction in the capacity for acidogenesis, and all three mutant strains did not survive an acid shock. According to biofilm and extracellular polysaccharides (EPS) formation analysis, all the mutant strains formed much less prolific biofilms with small amounts of EPS than wild-type UA159 when using L-ascorbate as the sole carbon source. Moreover, PCR analysis and quantitative real-time PCR revealed that sgaT, ptxA, ptxB, SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon. The transcription levels of these genes were all elevated in the presence of L-ascorbate, and the expression of ptxA gene decreased significantly once ptxB gene was knockout. The ptxA and ptxB genes are involved in the growth, aciduricity, acidogenesis, and formation of biofilms and EPS of S. mutans when L-ascorbate is the sole carbon source. In addition, the expression of ptxA is regulated by ptxB. ptxA, ptxB, and the upstream gene sgaT, the downstream genes SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon, and L-ascorbate is a potential inducer of the operon.

  4. 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

    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...... 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....

  5. Synthesis of a Hoechst 32258 Analogue Amino Acid Building Block for Direct Incorporation of a Fluorescent High-Affinity DNA Binding Motif into Peptides

    DEFF Research Database (Denmark)

    Harrit, Niels; Behrens, Carsten; Nielsen, P. E.

    2001-01-01

    The synthesis of a new versatile "Hoechst 33258-like" Boc-protected amino acid building block for peptide synthesis is described. It is demonstrated that this new ligand is an effective mimic of Hoechst 33258 in terms of DNA affinity and sequence specificity. Furthermore, this minor groove binder...

  6. Activating omega-6 polyunsaturated fatty acids and inhibitory purine nucleotides are high affinity ligands for novel mitochondrial uncoupling proteins UCP2 and UCP3

    Czech Academy of Sciences Publication Activity Database

    Žáčková, Markéta; Škobisová, Eva; Urbánková, Eva; Ježek, Petr

    2003-01-01

    Roč. 278, č. 23 (2003), s. 20761-20769 ISSN 0021-9258 R&D Projects: GA AV ČR IAA5011106; GA ČR GA301/02/1215; GA MŠk ME 389 Institutional research plan: CEZ:AV0Z5011922 Keywords : uncoupling protein-2 * polyunsaturated fatty acids * recombinant yeast expression Subject RIV: CE - Biochemistry Impact factor: 6.482, year: 2003

  7. Effects of x-irradiation induced loss of cerebellar granule cells on the synaptosomal levels and the high affinity uptake of amino acids

    International Nuclear Information System (INIS)

    Rohde, B.H.; Rea, M.A.; Simon, J.R.; McBride, W.J.

    1979-01-01

    Crude synaptosomal (P 2 ) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X-irradiation treatment and from the cerebella of control animals. In the P 2 fraction form control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2-fold higher than taurine. The content of taurine, GABA, glycine, and alanine were not changed by the X-irradiation treatment. The uptake of 1.0 micrometers-L-[ 3 H]glutamate and L-[ 3 H]aspartate was reduced approx 20% by X-irradiation treatment, whereas the uptake of 1.0 micrometers-[ 3 H]GABA and [ 3 H]taurine was unchanged. In a second study, the uptake of L-[ 3 H]glutamate, L-[ 3 H]aspartate and [ 3 H]GABA was measured using P 2 fractions obtained from the cerebella of rats in which the population of granule, stellate and basket cells had been reduced by X-irradiation treatment. The uptake of 1.0 micrometers-L-[ 3 H]glutamate, L-[ 3 H]aspartate and [ 3 H]GABA was significantly (P < 0.05) reduced to 57.68 and 59% respectively, of control values. The data are discussed in terms of glutamate being the excitatory neurotransmitter released from granule cells and GABA being the inhibitory neurotransmitter released from basket cells. (author)

  8. High affinity hemoglobin and Parkinson's disease.

    Science.gov (United States)

    Graham, Jeffrey; Hobson, Douglas; Ponnampalam, Arjuna

    2014-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain. Oxidative damage in this region has been shown to play an important role in the pathogenesis of this disease. Human neurons have been discovered to contain hemoglobin, with an increased concentration seen in the neurons of the SN. High affinity hemoglobin is a clinical entity resulting from mutations that create a functional increase in the binding of hemoglobin to oxygen and an inability to efficiently unload it to tissues. This can result in a number of metabolic compensatory changes, including an elevation in circulating hemoglobin and an increase in the molecule 2,3-diphosphoglycerate (2,3-DPG). Population based studies have revealed that patients with PD have elevated hemoglobin as well as 2,3-DPG levels. Based on these observations, we hypothesize that the oxidative damage seen in PD is related to an underlying high affinity hemoglobin subtype. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2015-07-15

    Mammalian colon harbours trillions of bacteria under physiological conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. In the present study we show that Slc5a8 (solute carrier gene family 5a, member 8), a Na(+)-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T-cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naive T-cells into immunosuppressive forkhead box P3(+) (FoxP3(+)) Tregs (regulatory T-cells) and suppress conversion of naive T-cells into pro-inflammatory interferon (IFN)-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T-cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T-cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fibre intake but not when dietary fibre intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fibre and mucosal immune system via the bacterial metabolite butyrate and that this transporter is a conditional tumour suppressor in colon linked to dietary fibre content. © 2015 Authors; published by Portland Press Limited.

  10. The yiaKLX1X2PQRS and ulaABCDEFG Gene Systems Are Required for the Aerobic Utilization of l-Ascorbate in Klebsiella pneumoniae Strain 13882 with l-Ascorbate-6-Phosphate as the Inducer▿

    OpenAIRE

    Campos, Evangelina; de la Riva, Lucia; Garces, Fernando; Giménez, Rosa; Aguilar, Juan; Baldoma, Laura; Badia, Josefa

    2008-01-01

    The capacity to both ferment and oxidize l-ascorbate has been widely documented for a number of enteric bacteria. Here we present evidence that all the strains of Klebsiella pneumoniae tested in this study ferment l-ascorbate using the ula regulon-encoded proteins. Under aerobic conditions, several phenotypes were observed for the strains. Our results showed that the yiaK-S system is required for this aerobic metabolic process. Gel shift experiments performed with UlaR and YiaJ and probes cor...

  11. Formulation and characterization of a multiple emulsion containing 1% L-ascorbic acid

    Directory of Open Access Journals (Sweden)

    Naveed Akhtar

    2010-04-01

    Full Text Available The purpose of the study was to prepare a stable multiple emulsion containing a skin anti-aging agent and using paraffin oil. Vitamin C, was incorporated into the inner aqueous phase of water-in-oil-in-water (w/o/w multiple emulsion at a concentration of 1%. Multiple emulsion was prepared by two step method. Stability studies were performed at different accelerated conditions, i.e. 8 oC (in refrigerator, 25 oC (in oven, 40 oC (in oven, and 40 oC at 75% RH (in stability cabin for 28 days to predict the stability of formulations. Different parameters, namely pH, globule size, electrical conductivity and effect of centrifugation (simulating gravity were determined during stability studies. Data obtained was evaluated statistically using ANOVA two way analyses and LSD tests. Multiple emulsion formulated was found to be stable at lower temperatures (i.e. 8 and 25 oC for 28 days. No phase separation was observed in the samples during stability testing. It was found that there was no significant change (p > 0.05 in globule sizes in most of the samples kept at various conditions. Insignificant changes (p > 0.05 in both pH and conductivity values were determined for the samples kept at 8, 40, and 40 oC at 75% RH, throughout the study period. Further studies are needed to formulate more stable emulsions with other emulsifying agents.

  12. Improvement of 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid ...

    African Journals Online (AJOL)

    Methods: The production of AA-2G using UR or ultrasonic radiation with ... and 55 °C increased by 11.9 and 4.8 %, respectively, with a reduction in process time of 41.7 and 75 %, ... industry [1]. ... enzymatic transglycosylation reactions have.

  13. Study of L-ascorbic acid (vitamin C)/H 2O mixture across glass transition

    Science.gov (United States)

    Migliardo, F.; Branca, C.; Faraone, A.; Magazù, S.; Migliardo, P.

    2001-07-01

    In this paper, we report quasi elastic neutron scattering (QENS) spectra of vitamin C aqueous solutions, obtained using MIBEMOL spectrometer (LLB). The main purpose of this work is to characterize the relaxational and vibrational properties of the Vitamin C/H 2O system below and above the glass transition temperature by analysing the low-frequency neutron scattering spectra. The determination of the relative weight of vibrational over relaxational contributions allows to get information on the fragility degree of this peculiar hydrogen-bond system.

  14. L-ascorbic acid losses in Kenyan vegetables during cooking as ...

    African Journals Online (AJOL)

    The effect of the sharpness of the knife on the loss of L-AA was studied with spinach. It was found that more loss of L-AA occurred when a blunt (edge thickness 0.08 cm) knife was used for cutting the vegetables than when a sharp knife (edge thickness 0.04 cm) was used during cooking. L-AA was also determined when ...

  15. L-ascorbate attenuates methamphetamine neurotoxicity through enhancing the induction of endogenous heme oxygenase-1.

    Science.gov (United States)

    Huang, Ya-Ni; Wang, Jiz-Yuh; Lee, Ching-Tien; Lin, Chih-Hung; Lai, Chien-Cheng; Wang, Jia-Yi

    2012-12-01

    Methamphetamine (METH) is a drug of abuse which causes neurotoxicity and increased risk of developing neurodegenerative diseases. We previously found that METH induces heme oxygenase (HO)-1 expression in neurons and glial cells, and this offers partial protection against METH toxicity. In this study, we investigated the effects of l-ascorbate (vitamin C, Vit. C) on METH toxicity and HO-1 expression in neuronal/glial cocultures. Cell viability and damage were evaluated by 3-(4,5-dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release, respectively. Neuronal and glial localization of HO-1 were identified by double immunofluorescence staining. Reactive oxygen species (ROS) production was measured using the fluorochrome 2',7'-dichlorofluorescin diacetate. HO-1 mRNA and protein expression were examined by RT-qPCR and Western blotting, respectively. Results show that Vit. C induced HO-1 mRNA and protein expressions in time- and concentration-dependent manners. Inhibition of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK) significantly blocked induction of HO-1 by Vit. C. HO-1 mRNA and protein expressions were significantly elevated by a combination of Vit. C and METH, compared to either Vit. C or METH alone. Pretreatment with Vit. C enhanced METH-induced HO-1 expression and attenuated METH-induced ROS production and neurotoxicity. Pharmacological inhibition of HO activity abolished suppressive effects of Vit. C on METH-induced ROS production and attenuated neurotoxicity. We conclude that induction of HO-1 expression contributes to the attenuation of METH-induced ROS production and neurotoxicity by Vit. C. We suggest that HO-1 induction by Vit. C may serve as a strategy to alleviate METH neurotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. L-Ascorbate attenuates methamphetamine neurotoxicity through enhancing the induction of endogenous heme oxygenase-1

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ya-Ni [Department of Nursing, Hsin Sheng College of Medical Care and Management, Taoyuan, Taiwan (China); Wang, Jiz-Yuh [Department of Neurology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lee, Ching-Tien [Department of Nursing, Hsin Sheng College of Medical Care and Management, Taoyuan, Taiwan (China); Lin, Chih-Hung [Graduate Institute of Medical Sciences and Department of Physiology, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Lai, Chien-Cheng [Far Eastern Memorial Hospital, Department of Surgery, Taipei, Taiwan (China); Wang, Jia-Yi, E-mail: jywang2010@tmu.edu.tw [Graduate Institute of Medical Sciences and Department of Physiology, College of Medicine, Taipei Medical University, Taipei, Taiwan (China)

    2012-12-01

    Methamphetamine (METH) is a drug of abuse which causes neurotoxicity and increased risk of developing neurodegenerative diseases. We previously found that METH induces heme oxygenase (HO)-1 expression in neurons and glial cells, and this offers partial protection against METH toxicity. In this study, we investigated the effects of L-ascorbate (vitamin C, Vit. C) on METH toxicity and HO-1 expression in neuronal/glial cocultures. Cell viability and damage were evaluated by 3-(4,5-dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release, respectively. Neuronal and glial localization of HO-1 were identified by double immunofluorescence staining. Reactive oxygen species (ROS) production was measured using the fluorochrome 2′,7′-dichlorofluorescin diacetate. HO-1 mRNA and protein expression were examined by RT-qPCR and Western blotting, respectively. Results show that Vit. C induced HO-1 mRNA and protein expressions in time- and concentration-dependent manners. Inhibition of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK) significantly blocked induction of HO-1 by Vit. C. HO-1 mRNA and protein expressions were significantly elevated by a combination of Vit. C and METH, compared to either Vit. C or METH alone. Pretreatment with Vit. C enhanced METH-induced HO-1 expression and attenuated METH-induced ROS production and neurotoxicity. Pharmacological inhibition of HO activity abolished suppressive effects of Vit. C on METH-induced ROS production and attenuated neurotoxicity. We conclude that induction of HO-1 expression contributes to the attenuation of METH-induced ROS production and neurotoxicity by Vit. C. We suggest that HO-1 induction by Vit. C may serve as a strategy to alleviate METH neurotoxicity. -- Highlights: ► Besides the anti-oxidant effect, Vit. C also induces HO-1 expression in brain cells. ► Vit. C reduces METH neurotoxicity and ROS production by

  17. L-Ascorbate attenuates methamphetamine neurotoxicity through enhancing the induction of endogenous heme oxygenase-1

    International Nuclear Information System (INIS)

    Huang, Ya-Ni; Wang, Jiz-Yuh; Lee, Ching-Tien; Lin, Chih-Hung; Lai, Chien-Cheng; Wang, Jia-Yi

    2012-01-01

    Methamphetamine (METH) is a drug of abuse which causes neurotoxicity and increased risk of developing neurodegenerative diseases. We previously found that METH induces heme oxygenase (HO)-1 expression in neurons and glial cells, and this offers partial protection against METH toxicity. In this study, we investigated the effects of L-ascorbate (vitamin C, Vit. C) on METH toxicity and HO-1 expression in neuronal/glial cocultures. Cell viability and damage were evaluated by 3-(4,5-dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release, respectively. Neuronal and glial localization of HO-1 were identified by double immunofluorescence staining. Reactive oxygen species (ROS) production was measured using the fluorochrome 2′,7′-dichlorofluorescin diacetate. HO-1 mRNA and protein expression were examined by RT-qPCR and Western blotting, respectively. Results show that Vit. C induced HO-1 mRNA and protein expressions in time- and concentration-dependent manners. Inhibition of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK) significantly blocked induction of HO-1 by Vit. C. HO-1 mRNA and protein expressions were significantly elevated by a combination of Vit. C and METH, compared to either Vit. C or METH alone. Pretreatment with Vit. C enhanced METH-induced HO-1 expression and attenuated METH-induced ROS production and neurotoxicity. Pharmacological inhibition of HO activity abolished suppressive effects of Vit. C on METH-induced ROS production and attenuated neurotoxicity. We conclude that induction of HO-1 expression contributes to the attenuation of METH-induced ROS production and neurotoxicity by Vit. C. We suggest that HO-1 induction by Vit. C may serve as a strategy to alleviate METH neurotoxicity. -- Highlights: ► Besides the anti-oxidant effect, Vit. C also induces HO-1 expression in brain cells. ► Vit. C reduces METH neurotoxicity and ROS production by

  18. Susceptibility to endotoxin induced uveitis is not reduced in mice deficient in BLT1, the high affinity leukotriene B4 receptor

    OpenAIRE

    Smith, J R; Subbarao, K; Franc, D T; Haribabu, B; Rosenbaum, J T

    2004-01-01

    Aim: To investigate the role of arachidonic acid derived chemotactic factor, LTB4, in the development of endotoxin induced uveitis (EIU), using mice deficient in the BLT1 gene which encodes the high affinity LTB4 receptor.

  19. 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

  20. 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. Screen...

  1. Molecular basis of a high affinity murine interleukin-5 receptor.

    OpenAIRE

    Devos, R; Plaetinck, G; Van der Heyden, J; Cornelis, S; Vandekerckhove, J; Fiers, W; Tavernier, J

    1991-01-01

    The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by...

  2. Overproduction, crystallization and preliminary X-ray analysis of the putative l-ascorbate-6-phosphate lactonase UlaG from Escherichia coli

    International Nuclear Information System (INIS)

    Garces, Fernando; Fernández, Francisco J.; Pérez-Luque, Rosa; Aguilar, Juan; Baldomà, Laura; Coll, Miquel; Badía, Josefa; Vega, M. Cristina

    2007-01-01

    UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in E. coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized in a monoclinic space group. Crystals were obtained by the sitting-drop vapour-diffusion method at 293 K. A data set diffracting to 3 Å resolution was collected from a single crystal at 100 K. UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å 3 Da −1 and a solvent content of 43%

  3. L-dehydroascorbic acid can substitute L-ascorbic acid as dietary vitamin C source in guinea pigs

    DEFF Research Database (Denmark)

    Frikke-Schmidt, Henriette Rønne; Tveden-Nyborg, Pernille; Lykkesfeldt, Jens

    2016-01-01

    products prone to oxidation-was studied. Our aim was to compare tissue accumulation of vitamin C in guinea pigs receiving different oral doses of either ascorbate or DHA. In all tissues tested (plasma, liver, spleen, lung, adrenal glands, kidney, muscle, heart, and brain), only sporadic differences......, we continued to explore the ability to recycle DHA in blood, liver and intestine in guinea pigs, rats and mice. These investigations revealed that guinea pigs have similar recycling capacity in red blood cells as observed in humans, while rats and mice do not have near the same ability to reduce DHA...... in erythrocytes. In liver and intestinal homogenates, guinea pigs also showed a significantly higher ability to recycle DHA compared to rats and mice. These data demonstrate that DHA in guinea pigs-as in humans-is almost as effective as ascorbate as vitamin C source when it comes to taking up and storing vitamin...

  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. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition.

    Directory of Open Access Journals (Sweden)

    Yves Nominé

    Full Text Available Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15 derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F was equally well tolerated as the wild type glutamine (Q at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv-peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology. Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.

  6. Effect of Co-60 gamma radiation on optical, dielectric and mechanical properties of strontium L-ascorbate hexahydrate NLO crystal

    Science.gov (United States)

    Dileep, M. S.; Suresh Kumar, H. M.

    2018-04-01

    A potentially useful nonlinear optical semi-organic single crystal of strontium L-ascorbate hexahydrate (SLAH) was grown by solution growth slow evaporation technique at room temperature. The grown crystal is semi transparent, yellowish in color with monoclinic crystal system having space group P21 and is stable up to 198 °C. Further, SLAH crystals were irradiated with gamma rays produced by 60Co with different doses of 10 KGy, 30 KGy and 50 KGy at room temperature and then studied the effect of gamma-rays on dielectric properties, optical absorption, microhardness and SHG efficiency. The absorption study reveals that the absorbance of the grown crystal is appeared to be low throughout the visible region with a lower cutoff wavelength of 277 nm and these parameters are not affected upon gamma irradiation. The luminescence intensity of the crystal is also not affected by the irradiation. There is noticeable changes were observed in dielectric properties and hardness of the materials for different doses of gamma irradiation. The second harmonic generation (SHG) efficiency of the grown crystal is 0.54 times that of the KDP crystal and is decreased moderately by increasing the dosage of gamma irradiation.

  7. 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

    2017-07-17

    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 (K d =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.

  8. 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.

  9. 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.

  10. L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress, Autophagy, and Apoptosis.

    Science.gov (United States)

    Huang, Ya-Ni; Yang, Ling-Yu; Wang, Jing-Ya; Lai, Chien-Cheng; Chiu, Chien-Tsai; Wang, Jia-Yi

    2017-01-01

    Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.

  11. 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.

  12. Characterization of high-affinity (/sup 3/H)ouabain binding in the rat central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Hauger, R.; Luu, H.M.; Meyer, D.K.; Goodwin, F.K.; Paul, S.M.

    1985-06-01

    The characteristics of (/sup 3/H)ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of high-affinity binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. (/sup 3/H)Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific (/sup 3/H)ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for (/sup 3/H)ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal (/sup 3/H)ouabain binding was examined. Kainic acid lesions of the striatum reduced (/sup 3/H)ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the high-affinity (/sup 3/H)ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of (/sup 3/H)ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.

  13. Fc receptor-γ subunits with both polar or non-polar amino acids at position of T22 are capable of restoring surface expression of the high-affinity IgE receptor and degranulation in γ subunit-deficient rat basophilic leukemia cells

    Czech Academy of Sciences Publication Activity Database

    Rashid, A.; Housden, J.E.; Helm, B.A.; Dráber, Petr

    2013-01-01

    Roč. 53, č. 3 (2013), s. 270-273 ISSN 0161-5890 R&D Projects: GA MŠk LD12073; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA ČR(CZ) GBP302/12/G101; GA MŠk 1M0506 Grant - others:European Cooperation in Science and Technology(XE) BM1007; AV ČR(CZ) M200520901 Institutional research plan: CEZ:AV0Z50520514 Keywords : allergy * high-affinity IgE receptor * plasma membrane * transmembrane signaling * 3-helix assembly model Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.003, year: 2013

  14. Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts.

    Science.gov (United States)

    Yañez, Fernando; Chianella, Iva; Piletsky, Sergey A; Concheiro, Angel; Alvarez-Lorenzo, Carmen

    2010-02-05

    This work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA.HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross-linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA.HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g(-1) (i.e., 170 mg g(-1)) under flow

  15. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    International Nuclear Information System (INIS)

    Miles, L.A.; Plow, E.F.

    1986-01-01

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [ 125 I]EDP I, [ 125 I]Glu-plasminogen, and [ 125 I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [ 125 I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. α 2 -Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [ 125 I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

  16. 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...... 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...

  17. Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms

    Science.gov (United States)

    McQuaid, Jeffrey B.; Kustka, Adam B.; Oborník, Miroslav; Horák, Aleš; McCrow, John P.; Karas, Bogumil J.; Zheng, Hong; Kindeberg, Theodor; Andersson, Andreas J.; Barbeau, Katherine A.; Allen, Andrew E.

    2018-03-01

    In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton. Although most dissolved iron in the marine environment is complexed with organic molecules, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts, including the ferric iron-concentrating protein ISIP2A, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine environmental genomic datasets, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

  18. 99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor

    International Nuclear Information System (INIS)

    Lane, Stephanie R.; Veerendra, Bhadrasetty; Rold, Tammy L.; Sieckman, Gary L.; Hoffman, Timothy J.; Jurisson, Silvia S.; Smith, Charles J.

    2008-01-01

    Introduction: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. 99m Tc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. Methods: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [ 99m Tc(H 2 O) 3 (CO) 3 ] + . The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy ( 1 H and 13 C). DTMA was conjugated to H 2 N-(X)-BBN(7-14)NH 2 , where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH 2 conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. Results: The new conjugates were radiolabeled with [ 99m Tc(H 2 O) 3 (CO) 3 ] + produced via Isolink radiolabeling kits to produce [ 99m Tc(CO) 3 -DTMA-(X)-BBN(7-14)NH 2 ]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. Conclusions: [ 99m Tc(CO) 3 -DTMA-(X)-BBN(7-14)NH 2 ] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes

  19. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  20. Endothelial targeting of high-affinity multivalent polymer nanocarriers directed to intercellular adhesion molecule 1.

    Science.gov (United States)

    Muro, Silvia; Dziubla, Thomas; Qiu, Weining; Leferovich, John; Cui, Xiumin; Berk, Erik; Muzykantov, Vladimir R

    2006-06-01

    Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (approximately 200 nm diameter spheres carrying approximately 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 approximately 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax approximately 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd approximately 80 pM versus approximately 8 nM) in cell culture and, probably because of this factor, higher value (185.3 +/- 24.2 versus 50.5 +/- 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 +/- 10.9 versus 2.1 +/- 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

  1. Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

    Directory of Open Access Journals (Sweden)

    Johan Nilvebrant

    Full Text Available The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

  2. 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

  3. ISOLATION AND CHARACTERIZATION OF THE HIGH-AFFINITY K+-TRANSLOCATING ATPASE FROM RHODOBACTER-SPHAEROIDES

    NARCIS (Netherlands)

    ABEE, T; SIEBERS, A; ALTENDORF, K; KONINGS, WN

    1992-01-01

    Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K+-stimulated and Mg2+-stimulated ATPase was purified from membranes of these cells by solubilization with

  4. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase

    DEFF Research Database (Denmark)

    Fischer, R; Julsgart, J; Berchtold, M W

    1998-01-01

    M-binding peptide derived from the p110gamma isoform interacts with CaM in a calcium-dependent way. Using gel shift analysis and fluorescence spectrophotometry we discovered that the peptide forms a high affinity complex with CaM. Titration experiments using dansylated CaM gave an affinity constant of 5 n...

  5. The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

    Science.gov (United States)

    Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

    2017-08-01

    The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. 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. © 2012 John Wiley & Sons A/S.

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

    Energy Technology Data Exchange (ETDEWEB)

    Denardo, Sally J.; Denardo, Gerald L.; Balhorn, Rodney L.

    2018-02-06

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

  8. 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.

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

    OpenAIRE

    Milton, N G

    1999-01-01

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

  10. 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.

  11. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. 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.

  13. Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

    International Nuclear Information System (INIS)

    Ross, S.B.

    1987-01-01

    3 H-alaproclate, a selective 5 h ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K D -=3 nM) and large capacity (B max about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the 3 H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3 H-alaproclate binding with the same, high affinity (K i =3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3 H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed. (author)

  14. Proadifen-sensitive high affinity binding of /sup 3/H-alaproclate to liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B.

    1987-01-01

    /sup 3/H-alaproclate, a selective 5/sub h/ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K/sub D/-=3 nM) and large capacity (B/sub max/ about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the /sup 3/H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the /sup 3/H-alaproclate binding with the same, high affinity (K/sub i/=3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced /sup 3/H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.

  15. Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

    Science.gov (United States)

    Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Thermal degradation kinetics of ascorbic acid, thiamine and riboflavin in rosehip (Rosa canina L nectar

    Directory of Open Access Journals (Sweden)

    Çetin KADAKAL

    2017-10-01

    Full Text Available Abstract In this paper, the loss of L-ascorbic acid, thiamine and riboflavin in rosehip nectar with the heating periods (0, 5, 10, 15, 20 and 30 min at temperatures ranging from 70 to 95 °C is analyzed and experimental results are presented. Firstly, dried rosehip fruits were processed to rosehip nectar and then thermal treatment is performed. Liquid chromatographic (HPLC method was used for the analysis of the contents of L-ascorbic acid, thiamine and riboflavin and examined compounds are thoroughly separated within 25 min. During thermal processing, degradation of L-ascorbic acid, thiamine and riboflavin in rosehip nectar were fitted to a first-order reaction kinetic model. Arrhenius relationship was used for the description of temperature dependence of reaction. Activation energies for L-ascorbic acid, thiamine and riboflavin between 70 to 95 ºC were found to be 55.30, 36.38 and 37.15 kJ/mol, respectively. To the best of the author’s knowledge, due to lack of study on the thermal degradation of L-ascorbic acid, thiamine and riboflavin in rosehip nectar, this manuscript will be the first reported study to enable future analysis.

  17. Changes in medium radioactivity and composition accompany high-affinity uptake of glutamate and aspartate by mouse brain slices

    International Nuclear Information System (INIS)

    Latzkovits, L.; Neidle, A.; Lajtha, A.

    1984-01-01

    In measurements of high affinity transport in tissue slices, the incubation medium is often treated as an ''infinitely large pool''. External substrate concentrations, even at the micromolar level, are assumed to be constant and metabolic interactions between tissue and medium are neglected. In the present report we describe experiments in which glutamic and aspartic acid uptake by mouse brain slices were studied using techniques that could test these assumptions. Cerebral hemispheres were cut into 0.1 mm sections and about 90 mg of tissue incubated in 10 ml of oxygenated medium. After 45 minutes of equilibration, radioactive substrates were added and the concentrations and specific activities of the amino acids and their metabolites in the medium were determined. During the first 10 min following substrate addition, rapid decreases in glutamic and aspartic acid concentrations in the medium were accompanied by large decreases in specific activity caused by the continuous release of these amino acids from the tissue. In addition, extensive conversion of both substrates to glutamine and the preferential accumulation of this metabolite, in the medium, was found. These results demonstrate that metabolism and release occur simultaneously with uptake during transport experiments in vitro and that these processes can take place in specific tissue compartments. It is therefore necessary to measure the tissue and medium concentration levels of amino acids along with their radioactivity in such experiments, since all three processes (transport, metabolism, and compartmentation) are interrelated in the clearance of amino acids from the incubation medium and probably from the extracellular spaces in vivo as well

  18. High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

    DEFF Research Database (Denmark)

    Hansen, Mads E; Bentin, Thomas; Nielsen, Peter E

    2009-01-01

    While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes-mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine subs...

  19. Design, Synthesis, and Biological Evaluation of Small, High-Affinity Siglec-7 Ligands: Toward Novel Inhibitors of Cancer Immune Evasion.

    Science.gov (United States)

    Prescher, Horst; Frank, Martin; Gütgemann, Stephan; Kuhfeldt, Elena; Schweizer, Astrid; Nitschke, Lars; Watzl, Carsten; Brossmer, Reinhard

    2017-02-09

    Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar K d values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds.

  20. Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

    The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A 1 and A 2 adenosine binding sites. The ligands used were 2-chloro(8- 3 H) adenosine and N 6 -(adenine-2, 8- 3 H) cyclohexayladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The kinetic constant determinations from binding studies of the adenosine receptor are presented. Utilizing tritium-cyclohexyladenosine as ligand the authors observed evidence for a high affinity binding site in human brain cortical membranes with a kd of 5 nM

  1. 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...... demonstrated high affinity binding and selectivity for SERT (K i = 3 nM). Visualization of SERT, using confocal laser scanning microscopy, validated compound 14 as a novel tool for studying SERT expression and distribution in living cells....

  2. High affinity binding of [3H]cocaine to rat liver microsomes

    International Nuclear Information System (INIS)

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

    1988-01-01

    ] 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 [ 3 H]cocaine binding. On the other hand, chronic administration of cocaine reduced [ 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 [ 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 [ 3 H]cocaine binding to liver with a different rank order of potency than their displacement of [ 3 H]cocaine binding to striatum. This high affinity [ 3 H]cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  3. 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.

  4. High affinity, ligand specific uptake of complexed copper-67 by brain tissue incubated in vitro

    International Nuclear Information System (INIS)

    Barnea, A.; Hartter, D.E.

    1987-01-01

    Copper is an essential metal that is highly concentrated in the brain. The blood, the sole source of tissue Cu, contains 16-20 μM Cu, of which >95% is complexed to proteins and 2 was 10 times greater than that of CuAlbumin or Cu(II). Within the range of 0.2-150μM Cu, multiple uptake sites for CuHis were apparent. Increasing the molar ratio of His:Cu had a differential effect on Cu uptake: enhancing uptake at [Cu] 1 μM. Thus, using a His:Cu ratio of 1000, they observed a high affinity process exhibiting saturating and half saturating values of 5 μM and 1.5 μM Cu, respectively; using a His:Cu ratio of 2, they observed a low affinity process exhibiting saturating and half-saturating values of 100 μM and 40 μM Cu, respectively. Both processes required thermic but not metabolic energy, suggestive of facilitated diffusion. Considering the blood brain barrier for proteins, CuHis appears to be the major substrate for Cu uptake by neuronal tissue. They demonstrate the existence of a ligand specific, high affinity (apparent Km about 1.5 μM Cu) uptake process for CuHis in the brain, operative at the physiological concentration range of CuHis and histidine

  5. High-Affinity Accumulation of Chloroquine by Mouse Erythrocytes Infected with Plasmodium berghei

    Science.gov (United States)

    Fitch, Coy D.; Yunis, Norman G.; Chevli, Rekha; Gonzalez, Yolanda

    1974-01-01

    Washed erythrocytes infected with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) P. berghei were used in model systems in vitro to study the accumulation of chloroquine with high affinity. The CS model could achieve distribution ratios (chloroquine in cells: chloroquine in medium) of 100 in the absence of substrate. 200—300 in the presence of 10 mM pyruvate or lactate, and over 600 in the presence of 1 mM glucose or glycerol. In comparable studies of the CR model, the distribution ratios were 100 in the absence of substrate and 300 or less in the presence of glucose or glycerol. The presence of lactate stimulated chloroquine accumulation in the CR model, whereas the presence of pyruvate did not. Lactate production from glucose and glycerol was undiminished in the CR model, and ATP concentrations were higher than in the CS model. Cold, iodoacetate, 2,4-dinitrophenol, or decreasing pH inhibited chloroquine accumulation in both models. These findings demonstrate substrate involvement in the accumulation of chloroquine with high affinity. In studies of the CS model, certain compounds competitively inhibited chloroquine accumulation, while others did not. This finding is attributable to a specific receptor that imposes structural constraints on the process of accumulation. For chloroquine analogues, the position and length of the side chain, the terminal nitrogen atom of the side chain, and the nitrogen atom in the quinoline ring are important determinants of binding to this receptor. PMID:4600044

  6. High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

    International Nuclear Information System (INIS)

    Lach, E.; Trifilieff, A.; Landry, Y.; Gies, J.P.

    1991-01-01

    The binding of the radiolabeled bombesin analogue [ 125 I-Tyr 4 ]bombesin to guinea-pig lung membranes was investigated. Binding of [ 125 I-Tyr 4 ]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B max = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K D = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [ 125 I-Tyr 4 ]bombesin, neuromedin B and neuromedin C inhibited the binding of [ 125 I-Tyr 4 ]bombesin in an order of potencies as follows: [ 125 I-Tyr 4 ]bombesin > bombesin ≥ neuromedin C much-gt neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B

  7. Reconstitution of high affinity α2 adrenergic agonist binding by fusion with a pertussis toxin substrate

    International Nuclear Information System (INIS)

    Kim, M.H.; Neubig, R.R.

    1986-01-01

    High affinity α 2 adrenergic agonist binding is thought to occur via a coupling of the α 2 receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 μM phenoxybenzamine to block α 2 receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the α 2 agonist [ 3 H]UK 14,304 (UK) and the antagonist [ 3 H] yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain α 2 receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance [ 3 H] UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity α 2 agonist binding

  8. L-ascorbate attenuates the endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation and NF-κB translocation in cortical neurons/glia Cocultures.

    Directory of Open Access Journals (Sweden)

    Ya-Ni Huang

    Full Text Available In response to acute insults to the central nervous system, such as pathogen invasion or neuronal injuries, glial cells become activated and secrete inflammatory mediators such as nitric oxide (NO, cytokines, and chemokines. This neuroinflammation plays a crucial role in the pathophysiology of chronic neurodegenerative diseases. Endogenous ascorbate levels are significantly decreased among patients with septic encephalopathy. Using the bacterial endotoxin lipopolysaccharide (LPS to induce neuroinflammation in primary neuron/glia cocultures, we investigated how L-ascorbate (vitamin C; Vit. C affected neuroinflammation. LPS (100 ng/ml induced the expression of inducible NO synthase (iNOS and the production of NO, interleukin (IL-6, and macrophage inflammatory protein-2 (MIP-2/CXCL2 in a time-dependent manner; however, cotreatment with Vit. C (5 or 10 mM attenuated the LPS-induced iNOS expression and production of NO, IL-6, and MIP-2 production. The morphological features revealed after immunocytochemical staining confirmed that Vit. C suppressed LPS-induced astrocytic and microglial activation. Because Vit. C can be transported into neurons and glia via the sodium-dependent Vit. C transporter-2, we examined how Vit. C affected LPS-activated intracellular signaling in neuron/glia cocultures. The results indicated the increased activation (caused by phosphorylation of mitogen-activated protein kinases (MAPKs, such as p38 at 30 min and extracellular signal-regulated kinases (ERKs at 180 min after LPS treatment. The inhibition of p38 and ERK MAPK suppressed the LPS-induced production of inflammatory mediators. Vit. C also inhibited the LPS-induced activation of p38 and ERK. Combined treatments of Vit. C and the inhibitors of p38 and ERK yielded no additional inhibition compared with using the inhibitors alone, suggesting that Vit. C functions through the same signaling pathway (i.e., MAPK as these inhibitors. Vit. C also reduced LPS-induced Iκ

  9. The RCK1 high-affinity Ca2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels.

    Science.gov (United States)

    Hou, Shangwei; Xu, Rong; Heinemann, Stefan H; Hoshi, Toshinori

    2008-03-11

    Carbon monoxide (CO) is a lethal gas, but it is also increasingly recognized as a physiological signaling molecule capable of regulating a variety of proteins. Among them, large-conductance Ca(2+)- and voltage-gated K(+) (Slo1 BK) channels, important in vasodilation and neuronal firing, have been suggested to be directly stimulated by CO. However, the molecular mechanism of the stimulatory action of CO on the Slo1 BK channel has not been clearly elucidated. We report here that CO reliably and repeatedly activates Slo1 BK channels in excised membrane patches in the absence of Ca(2+) in a voltage-sensor-independent manner. The stimulatory action of CO on the Slo1 BK channel requires an aspartic acid and two histidine residues located in the cytoplasmic RCK1 domain, and the effect persists under the conditions known to inhibit the conventional interaction between CO and heme in other proteins. We propose that CO acts as a partial agonist for the high-affinity divalent cation sensor in the RCK1 domain of the Slo1 BK channel.

  10. Isolation and characterization of high affinity aptamers against DNA polymerase iota.

    Science.gov (United States)

    Lakhin, Andrei V; Kazakov, Andrei A; Makarova, Alena V; Pavlov, Yuri I; Efremova, Anna S; Shram, Stanislav I; Tarantul, Viacheslav Z; Gening, Leonid V

    2012-02-01

    Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

  11. Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

    International Nuclear Information System (INIS)

    Ogura, Kenji; Shiga, Takanori; Yokochi, Masashi; Yuzawa, Satoru; Burke, Terrence R.; Inagaki, Fuyuhiko

    2008-01-01

    The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity

  12. Haemoglobin Pierre-Benite--a high affinity variant associated with relative polycythaemia.

    Science.gov (United States)

    Beard, M E; Potter, H C; Spearing, R L; Brennan, S O

    2001-12-01

    This is the second reported example of Hb Pierre--Benite (beta90 Glu-->Asp). This mutation is associated with increased oxygen affinity and polycythaemia. No instability was found and there was no charge shift detected by cellulose acetate electrophoresis at pH 8.3. The mutation was however, clearly indicated by electrospray ionization mass spectrometry (ESI MS), which showed an abnormal beta chain with a 14 Da decrease in mass. Blood volume studies documented a relative rather than a true polycythaemia and this finding has been reported in at least two other high affinity haemoglobin variants--Hb Heathrow and Hb Rahere. This finding led to delay in diagnosis because high oxygen affinity variants are conventionally considered to cause a true polycythaemia.

  13. New immunogenic form for vasopressin: production of high-affinity antiserum and RIA for plasmatic AVP

    International Nuclear Information System (INIS)

    Rougon-Rappuzi, G.; Delaage, M.A.; Conte-Devolx, B.; Millet, Y.

    1977-01-01

    A highly sensitive and specific radioimmunoassay (RIA) for arginine-vasopressin (AVP) was developped and applied to the measurement of AVP in human plasma. High-affinity antivasopressin antibodies with limited association constant heterogeneity have been induced by immunizing rabbits with Lysine-vasopressine (LVP) coupled to a human immunoglobulin (IgA). Replacing air drying of acetone-petroleum ether extracts by lyophilisation increased significantly the yields of AVP. Equilibrium dialysis was used for separating bound and free antigen, thus reducing the total time required for the assay to 48 hours. Only 1 ml of plasma was required for routine determinations due to a sensitivity threshold better than 0.5 pg/ml. Plasma AVP levels of normal subjects and of patients with inappropriate ADH secretion (SIADH) were determined during different hydratation states and following nicotin of ethanol infusions. (orig.) [de

  14. Exploiting the high-affinity phosphonate-hydroxyapatite nanoparticle interaction for delivery of radiation and drugs

    International Nuclear Information System (INIS)

    Ong, Hooi Tin; Loo, Joachim S. C.; Boey, Freddy Y. C.; Russell, Stephen J.; Ma Jan; Peng, Kah-Whye

    2008-01-01

    Hydroxyapatite is biocompatible and used in various biomedical applications. Here, we generated hydroxyapatite nanoparticles (HNPs) of various sizes (40-200 nm) and demonstrated that they can be stably loaded with drugs or radioisotopes by exploiting the high-affinity HA-(poly)phosphonate interaction. Clinically available phosphonates, clodronate, and Tc-99m-methylene-diphosphonate (Tc-99m-MDP), were efficiently loaded onto HNPs within 15 min. Biodistribution of radiolabeled HNP-MDP-Tc99m in mice was monitored non-invasively using microSPECT-CT. Imaging and dosimetry studies indicated that the HNPs, regardless of size, were quickly taken up by Kupffer cells in the liver after systemic administration into mice. Clodronate loaded onto HNPs remained biologically active and were able to result in selective depletion of Kupffer cells. This method of drug or isotope loading on HA is fast and easy as it eliminates the need for additional surface modifications of the nanoparticles

  15. High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

    International Nuclear Information System (INIS)

    Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

    2014-01-01

    Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

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

    International Nuclear Information System (INIS)

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

    1985-01-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; [ 3 H]-5-HT). At concentrations in the range of 0.01 to 0.7 microM [ 3 H]-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM [ 3 H]-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of [ 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 [ 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 [ 3 H]-5-HT up to 44-fold at an external [ 3 H]-5-HT concentration of 10(-7) M. Inhibition of [ 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 [ 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 [ 3 H]-5-HT uptake

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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 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 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

  18. 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.

  19. A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

    Science.gov (United States)

    Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

    2003-01-01

    A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

  20. Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

    Science.gov (United States)

    Lai, H; Carino, M A

    1992-07-01

    Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

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

    OpenAIRE

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-01-01

    Zr(IV) can form phosphate and Zr(IV) (?PO3 2??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). Aft...

  2. 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.

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

    Science.gov (United States)

    Skottrup, Peter Durand

    2017-09-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for RgpB 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. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    Calligaro, D.; Eldefrawi, M.

    1986-01-01

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

  5. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

    Science.gov (United States)

    Turk, B.; Stoka, V.; Björk, I.; Boudier, C.; Johansson, G.; Dolenc, I.; Colic, A.; Bieth, J. G.; Turk, V.

    1995-01-01

    Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain. PMID:8528085

  6. 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

  7. Identification of a High Affinity Nucleocapsid Protein Binding Element from The Bovine Leukemia Virus Genome

    Science.gov (United States)

    Yildiz, F. Zehra; Babalola, Kathleen; Summers, Michael F.

    2012-01-01

    Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5′-untranslated region (5′-UTR) of the genome. Recent studies suggest that a major packaging determinant of Bovine Leukemia Virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5′-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (Kd = 136 ± 21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369- U399, Kd = 67 ± 8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism. PMID:22846919

  8. Identification of high-affinity calmodulin-binding proteins in rat liver

    International Nuclear Information System (INIS)

    Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

    1987-01-01

    The Ca 2+ -dependent binding of [ 125 I] calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca 2+ -dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca 2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver

  9. 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.

  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. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    Science.gov (United States)

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

  12. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Directory of Open Access Journals (Sweden)

    Béatrice Marquèze-Pouey

    Full Text Available Signaling mediated by the epidermal growth factor (EGF is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer. In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  13. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Science.gov (United States)

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  14. Specific, high affinity receptors for insulin-like growth factor II in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1988-01-01

    Rat renal glomeruli were isolated by a technique involving kidney perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving, and concentration over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components, while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabeled IGF-II, with 50% inhibition at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked with disuccinimidyl suberate to its receptor in rat renal glomeruli and a specific high mol wt (255,000) band could be identified on autoradiograms of dodecyl sulfate-polyacrylamide gels. [ 125 I]IGF-II binding and cross-linking to this band was inhibited by a polyclonal antibody against the type II IGF receptor. These results demonstrate for the first time that the isolated rat renal glomerulus contains a high affinity receptor for IGF-II

  15. 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.

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

    International Nuclear Information System (INIS)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta 9 tetrahydrocannabinol (delta 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'-Trimethylammonium-delta 8 THC (TMA) is a positively charged analog of delta- 8 THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [ 3 H]-5'-trimethylammonium-delta- 8 THC ([ 3 H]TMA) to rat neuronal membranes. [ 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 [ 3 H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

  17. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    International Nuclear Information System (INIS)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A.

    1990-01-01

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite

  18. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. (Indian Institute of Chemical Biology, Calcutta (India))

    1990-07-05

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

  19. Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

    International Nuclear Information System (INIS)

    Asermely, K.E.; O'Neill, J.J.

    1986-01-01

    The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37 0 C in buffers containing tritium choline (5 μM 0.124 μC/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10 -5 M compared with 50% inhibition on brain synaptosomes. At 5 x 10 -4 M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K + -ion (25 mM) were unsuccessful; tissue 3 H-choline uptake appeared to oscillate over a 30 min period

  20. Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

    Energy Technology Data Exchange (ETDEWEB)

    Asermely, K.E.; O' Neill, J.J.

    1986-03-01

    The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37/sup 0/C in buffers containing tritium choline (5 ..mu..M 0.124 ..mu..C/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10/sup -5/ M compared with 50% inhibition on brain synaptosomes. At 5 x 10/sup -4/ M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K/sup +/-ion (25 mM) were unsuccessful; tissue /sup 3/H-choline uptake appeared to oscillate over a 30 min period.

  1. Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation.

    Science.gov (United States)

    Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

    2011-03-07

    The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate.

  2. 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....

  3. UlaR activates expression of the ula operon in Streptococcus pneumoniae in the presence of ascorbic acid

    NARCIS (Netherlands)

    Afzal, Muhammad; Shafeeq, Sulman; Henriques-Normark, Birgitta; Kuipers, Oscar P

    In this study, the regulatory mechanism of the ula (utilization of l-ascorbic acid) operon, putatively responsible for transport and utilization of ascorbic acid in Streptococcus pneumoniae strain D39, is studied. β-Galactosidase assay data demonstrate that expression of the ula operon is increased

  4. Carbon-11-labelling of a novel, trishomocubane-derived, high affinity and selectivity DAT ligand

    International Nuclear Information System (INIS)

    Dolle, F.; Le Helleix, St.; Peyronneau, M.A.; Saba, W.; Tournier, N.; Valette, H.; Banister, S.; Kassiou, M.

    2011-01-01

    Complete text of publication follows: Objectives: Parkinson's disease, schizophrenia, attention deficit disorder and drug abuse are related to abnormalities within the brain's dopaminergic system. The neuronal dopamine transporter (DAT) plays a key role in regulating the synaptic concentration of dopamine and thus dopamine neurotransmission in the brain. Since the DAT can be considered as a marker of the integrity and number of the presynaptic striatal dopamine-producing neurons, considerable efforts have been spent in recent years on the design and development of DAT-selective radioligands for use in Positron Emission Tomography (PET) studies. Notably, the tropane PE2I and its fluorinated analogue LBT-999 were identified as having high affinity and selectivity for the DAT over the norepinephrine transporter (NET) and the serotonin transporter (SERT). Besides tropanes, only a few bicyclic frameworks, e.g. bicyclo[2.2.2]octanes, have delivered compounds with high affinity for the DAT. Recently, novel poly-carbocyclic DAT ligands with selectivity over the NET and the SERT were reported. The lead compound of this series (1, N-methyl-N-(3-fluoro) benzyl-pentacyclo[5.4.0.0 2, 6 .0 3, 10 .0 5, 9 ] undec-8-ylamine, Ki = 1.2 nM, ≥ 8300-fold selectivity over NET and SERT) was selected as a potential candidate for imaging the DAT with PET and isotopically labelled with carbon-11 using [ 11 C]methyl triflate. Methods: The trishomocubane derivatives 1 (reference) and 2 (precursor for labelling with carbon-11) were prepared from commercially available Cookson's diketone in 6 and 7 steps, respectively. Carbon-11 labelling of 1 was performed using a TRACERLab FX-C Pro synthesizer (GEMS) and comprises (1) trapping at -10 C of [ 11 C]MeOTf in acetone (0.4 mL) containing the nor-derivative 2 (0.6-0.9 mg, free base) and aq. 3N NaOH (8 μL); (2) heating at 110 C for 2 min; (3) concentration to dryness and taking up the residue in 1.0 mL of the HPLC mobile phase; (4) purification

  5. Blockage of High-Affinity Choline Transporter Increases Visceral Hypersensitivity in Rats with Chronic Stress

    Directory of Open Access Journals (Sweden)

    Chen Zhao

    2018-01-01

    Full Text Available Background. Visceral hypersensitivity is a common feature of irritable bowel syndrome. Cholinergic system involves in the development of visceral hypersensitivity, and high-affinity choline transporter (CHT1 is of crucial importance in choline uptake system. However, involvement of CHT1 in visceral hypersensitivity remains unknown. The research aimed to study the CHT1 expression in dorsal root ganglions (DRGs and the role of CHT1 in visceral hypersensitivity. Methods. Repetitive water avoidance stress (WAS was used to induce visceral hypersensitivity in rats. Colorectal distension (CRD was determined, and the abdominal withdrawal reflex (AWR and threshold intensity data were recorded to measure the visceral sensitivity. After intraperitoneal injection of hemicholinium-3 (HC-3, the specific inhibitor of CHT1, CRD data were also recorded. The CHT1 expression of DRGs was investigated by Western blotting, immunohistochemistry, and quantitative RT-PCR. Acetylcholine levels in the DRGs were detected by the assay kit. Results. Repetitive WAS increased the AWR score of CRD at high distension pressure and decreased the mean threshold of rats. The CHT1 expression and acetylcholine concentration of DRG were significantly increased in WAS rats. After the administration of HC-3, the AWR score in WAS group was significantly increased at higher distension pressure while the threshold intensity was significantly reduced compared to the normal saline group. Acetylcholine concentration was significantly lower than the normal saline rats. Conclusion. Our research firstly reports that CHT1 is overexpressed in noninflammatory visceral hypersensitivity, and blockage of CHT1 can enhance the visceral hypersensitivity. CHT1 may play an inhibitory role in visceral hypersensitivity.

  6. Blockage of High-Affinity Choline Transporter Increases Visceral Hypersensitivity in Rats with Chronic Stress

    Science.gov (United States)

    2018-01-01

    Background Visceral hypersensitivity is a common feature of irritable bowel syndrome. Cholinergic system involves in the development of visceral hypersensitivity, and high-affinity choline transporter (CHT1) is of crucial importance in choline uptake system. However, involvement of CHT1 in visceral hypersensitivity remains unknown. The research aimed to study the CHT1 expression in dorsal root ganglions (DRGs) and the role of CHT1 in visceral hypersensitivity. Methods Repetitive water avoidance stress (WAS) was used to induce visceral hypersensitivity in rats. Colorectal distension (CRD) was determined, and the abdominal withdrawal reflex (AWR) and threshold intensity data were recorded to measure the visceral sensitivity. After intraperitoneal injection of hemicholinium-3 (HC-3), the specific inhibitor of CHT1, CRD data were also recorded. The CHT1 expression of DRGs was investigated by Western blotting, immunohistochemistry, and quantitative RT-PCR. Acetylcholine levels in the DRGs were detected by the assay kit. Results Repetitive WAS increased the AWR score of CRD at high distension pressure and decreased the mean threshold of rats. The CHT1 expression and acetylcholine concentration of DRG were significantly increased in WAS rats. After the administration of HC-3, the AWR score in WAS group was significantly increased at higher distension pressure while the threshold intensity was significantly reduced compared to the normal saline group. Acetylcholine concentration was significantly lower than the normal saline rats. Conclusion Our research firstly reports that CHT1 is overexpressed in noninflammatory visceral hypersensitivity, and blockage of CHT1 can enhance the visceral hypersensitivity. CHT1 may play an inhibitory role in visceral hypersensitivity. PMID:29849603

  7. Characterization of high affinity [3H]triazolam binding in rat brain

    International Nuclear Information System (INIS)

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-01-01

    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 [ 3 H]TZ binding. Saturation studies showed a shift to lower affinity at 37 0 C (K/sub d/ = 0.25 +/- 0.01 nM at O 0 C; K/sub d/ = 1.46 +/- 0.03 nM at 37 0 C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0 0 C and 1001 +/- 43 fmoles/mg prot. at 37 0 C). Inhibition studies showed that [ 3 H]TZ binding displayed no GABA shift at 0 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 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 0 C. In Ro 15-1788/[ 3 H]TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on [ 3 H]TZ binding at both temperatures. In conclusion [ 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

  8. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms.

    Science.gov (United States)

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-04-17

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments' ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

  9. Two classes of astrocytes in the adult human and pig retina in terms of their expression of high affinity NGF receptor (TrkA).

    Science.gov (United States)

    Ruiz-Ederra, Javier; Hitchcock, Peter F; Vecino, Elena

    2003-02-13

    Astrocytes have been implicated in axon guidance and synaptic regeneration in the retina and these processes involve activation of the high affinity nerve growth factor receptor, known as the tyrosine kinase A (TrkA) receptor. The purpose of the present study was to characterize the expression of TrkA in astrocytes of the adult pig and human retina. To this end, sections of human and pig retinas were immunolabeled with a combination of antibodies to glial fibrillary acidic protein (GFAP) and TrkA. Our study revealed that most of the GFAP-positive cells express TrkA, whereas a rare, novel subpopulation of astrocytes was found to be devoid of TrkA. Our results support the idea that astrocytes play an important neurotrophic role in the retina.

  10. 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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 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 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. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

    International Nuclear Information System (INIS)

    Borgundvaag, B.; George, S.R.

    1985-01-01

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [ 3 H]-ATP to [ 3 H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC 50 values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC 50 values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D 2 dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table

  13. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  14. Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

    OpenAIRE

    Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

    1981-01-01

    We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

  15. Photoaffinity labeling of mammalian α1-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe

    International Nuclear Information System (INIS)

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.

    1984-01-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated α 1 -adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[ 125 I]iodophenyl)pentanoyl]-1-piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an α 1 -adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical α 1 -adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the α 1 -adrenergic receptor

  16. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    International Nuclear Information System (INIS)

    Orito, N; Umekage, S; Sakai, E; Tanaka, T; Kikuchi, Y; Sato, K; Kawauchi, S; Tanaka, H

    2012-01-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be K D = 2.25x10 -9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  17. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    International Nuclear Information System (INIS)

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-01-01

    Forskolin labelled with [ 3 H] bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg 2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins

  18. Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

    International Nuclear Information System (INIS)

    Penefsky, H.S.

    1985-01-01

    Incubation of [gamma- 32 P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma- 32 P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma- 32 P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide

  19. High-affinity small molecule-phospholipid complex formation: binding of siramesine to phosphatidicacid

    DEFF Research Database (Denmark)

    Khandelia, Himanshu

    2008-01-01

    , comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction of XPA ) 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 ( 80 × 106. An MD simulation on the siramesine:PA interaction...

  20. 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

  1. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay.

    Science.gov (United States)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen

    2017-01-01

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  2. Structural implications of hERG K+ channel block by a high-affinity minimally structured blocker

    Science.gov (United States)

    Helliwell, Matthew V.; Zhang, Yihong; El Harchi, Aziza; Du, Chunyun; Hancox, Jules C.; Dempsey, Christopher E.

    2018-01-01

    Cardiac potassium channels encoded by human ether-à-go-go–related gene (hERG) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block. PMID:29545312

  3. 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.

  4. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen (UMASS, MED); (Pfizer)

    2017-09-21

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  5. 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......)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [3H]5-HT uptake in COS-7 cells (Ki = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative...

  6. 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.

  7. High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

    Science.gov (United States)

    Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

    2015-01-01

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

  8. High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

    Science.gov (United States)

    Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

    2015-02-13

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

    Science.gov (United States)

    Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

    2017-05-13

    DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A.; Nathenson, Stanley G.; Guha, Chandan; Almo, Steven C.

    2017-03-01

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  11. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Directory of Open Access Journals (Sweden)

    Eszter Lázár-Molnár

    2017-03-01

    Full Text Available Programmed Cell Death-1 (PD-1 is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1 exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

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

    International Nuclear Information System (INIS)

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

    1982-01-01

    Specific binding of 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

  13. Radioiodsodestannylation. Convenient synthesis of a high affinity thromboxane A2/prostaglandin H2 receptor antagonist

    International Nuclear Information System (INIS)

    Mais, D.E.; Hamanaka, Nobuyuki

    1991-01-01

    Radioiodination of methyl-7-[(2R, 2S, 5R)-6,6-dimethyl-3-(4-trimethylstannylbenzenesulfononylamino3S) bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoate with [ 125 I] Na using a modification of the chloramine-T method in organic solvent is simple with high yields and site specific. The product, following hydrolysis of the ester, 7-[(2R, 2S, 3S, 5R)-6,6-dimethyl-3-(4[ 125 I]-iodobenzenesulfonylamino) bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid [( 125 I]-ISAP), was purified by HPLC. The high specific activity and specific binding will make the ligand a useful tool for the characterization of thromboxane A 2 /prostaglandin H 2 receptors. (author)

  14. A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

    Science.gov (United States)

    Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

    2012-10-12

    The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

  15. High affinity (3H) β-Alanine uptake by scar margins of ferric chloride-induced epileptogenic foci in rat isocortex

    International Nuclear Information System (INIS)

    Robitaille, Y.; Sherwin, A.

    1984-01-01

    Cortical astrocytes of normal mammalian brain are endowed with a high affinity uptake system for β-Alanine which is competitively inhibited by gamma aminobutyric acid (GABA), a neurotransmitter strongly implicated in epileptogenesis. The authors evaluated ( 3 H) β-Alanine uptake by reactive astrocytes proliferating within scar of epileptogenic foci induced in rat motor cortex by microinjections of 100 mM ferric chloride. Following in vitro incubation of scar tissue with ( 3 H) β-Alanine, ultrastructural morphometry of grain patterns at 5, 30 and 120 days post injection revealed early and significant grain count increases over astroglial processes, predominantly those related to perivascular glial end-feet. Astrocytic cell body and endothelial cell counts showed a more gradual and stepwise increase. Similar data were obtained by comparing visual and edited mean astrocytic grain counts. These results suggest that the enhanced uptake of reactive astrocytes may reflect a marked decrease of inhibitory GABAergic neurons within ferric chloride-induced scars. 7 figures, 1 table

  16. Measurement of binding of ascorbic acid to myrosinase by rate of dialysis

    International Nuclear Information System (INIS)

    Ohtsuru, Masaru; Hata, Tadao

    1975-01-01

    The activation mechanism of myrosinase by L-ascorbic acid depends on the slight conformational change of enzyme protein induced by ascorbic acid. Ascorbic acid binds to enzyme like Michaelis-complex, and then the value of Km had been evaluated to be 1 x 10 -3 M. The authors determined the binding constant and the number of binding sites using dialysis rate technique. Rate dialysis was carried out with a dialysis cell, and the ordinary cellophane tubing membrane was used. ( 14 C) - ascorbic acid was added, and counted by liquid scintillation counting. By the time course of two dialysis rate measurement with and without enzyme. The concentrations of free and bound ascorbic acids were counted. From the results, the enzyme was activated to the maximum level at 10 -3 M of ascorbic acid, and four molecules of ascorbic acid bound to the enzyme on Kd=0.1x10 -4 M. However, when more than 4 molecules of L-ascorbic acid bound to the enzyme, Kd increased to 0.9x10 -4 M, and L-ascorbic acid acted as an inhibitor. (Kubatake, H.)

  17. Indium-111 tropolone, a new high-affinity platelet label: preparation and evaluation of labeling parameters

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Rao, S.A.; Didisheim, P.

    1981-01-01

    Platelets were isolated with a new neutral, lipid-soluble metal complex of indium-111 and tropolone. Unlike oxine, which must be dissolved in ethyl alcohol, tropolone is soluble saline. Platelet labeling with In-111 tropolone can be performed in both acid-citrate-dextrose (ACD)-plasma and ACD-saline media within two hours' time. Labeling efficiency has been 80-90% in ACD-saline and 60-70% in the ACD-plasma medium. Optimum concentrations for the labeling of platelets with In-111 tropolone were 5 micrograms/ml in ACD-saline and 10 micrograms/ml in ACD-plasma, using a 15-min incubation at room temperature. A kit formulation for convenient routine preparation of In-111-labeled platelets has been developed. Seven parameters of platelet labeling were studied: concentration of tropolone, citrate, plasma proteins, and calcium ions; also platelet density, temperature, and pH of incubation medium. Their effects on the mechanism of platelet labeling with lipid-soluble tracers are discussed

  18. Design of a peptidic turn with high affinity for HgII

    DEFF Research Database (Denmark)

    Pires, Sara; Habjanic, Jelena; Sezer, Murat

    2012-01-01

    A four amino acid peptide containing the ß-turn template dPro-Pro in the middle and two cysteines (Cys) in the terminal positions (CdPPC) has been synthesized and its mercury(II) coordination properties studied using different spectroscopic methods. The UV-vis, CD, (199m)Hg PAC, and Raman...... spectroscopic studies indicate the binding of Hg(II) to the two Cys, forming the dithiolatemercury(II) complex Hg(CdPPC). Electrospray ionization mass spectrometry corroborates the 1:1 complex formation. A log K = 40.0 was determined for the formation constant of the Hg(CdPPC) complex using competition...... potentiometric studies. Replacement of the dPro-Pro motif by a Pro-Pro unit generated a peptide (CPPC) capable of forming a similar species [Hg(CPPC)] but showing a lower affinity for Hg(II) (at least 3-3.5 orders of magnitude lower). The introduction of the dPro-Pro motif is crucial to induce the folding...

  19. High-affinity uranyl-specific antibodies suitable for cellular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C. [CEA Valrho, DSV, IBEB, Serv Biochim et Toxicol Nucl, F-30207 Bagnols Sur Ceze (France)

    2008-07-01

    Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO{sub 2}{sup 2+}-DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO{sub 2}{sup 2+}-DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K{sub D} = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO{sub 2}{sup 2+}, Fe{sup 3+}, Zn{sup 2+}, Cu{sup 2+}, and Ca{sup 2+}) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO{sub 2}{sup 2+} equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

  20. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

    Science.gov (United States)

    Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

    2006-12-21

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

  1. High-affinity uranyl-specific antibodies suitable for cellular imaging

    International Nuclear Information System (INIS)

    Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C.

    2008-01-01

    Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO 2 2+ -DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO 2 2+ -DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K D = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO 2 2+ , Fe 3+ , Zn 2+ , Cu 2+ , and Ca 2+ ) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO 2 2+ equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

  2. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...... that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level....

  3. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    Science.gov (United States)

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. 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 A 520nm / A 650nm 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.

  5. 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.

  6. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    International Nuclear Information System (INIS)

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-01-01

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 0 C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17β-[ 3 H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins

  7. 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

    2010-02-01

    Full Text Available 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.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.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.

  8. 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.

  9. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

    Science.gov (United States)

    Honey, Denise M.; Best, Annie; Qiu, Huawei

    2018-01-01

    ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

  10. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    International Nuclear Information System (INIS)

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

    1990-01-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

  11. A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

    Science.gov (United States)

    Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

    2014-01-01

    Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

  12. Comparison of high affinity binding of 3H-proadifen and 3H-(-)-cocaine t rat liver membranes

    International Nuclear Information System (INIS)

    Ross, S.B.

    1995-01-01

    The characteristics of the binding of 3 H-proadifen to rat liver membranes were studied and compared to those of 3 H-cocaine. It was found that 3 H-proadifen was bound reversibly with high affinity (K D =1.8±0.5 nM) and large capacity (B max =2010±340 pmol/g wet tissue) to liver membranes. The corresponding values for the 3 H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of 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 μM CdCl 2 in the incubation buffer it was possible to differentiate between two 3 H-cocaine binding sites with K d values of 1.6 and 7.7 nM and B max values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of 3 H-proadifen and 3 H-cocaine inhibited the binding of 3 H-proadifen (IC 50 =10 nM) and proadifen that of 3 H-cocaine (IC 50 =1 nM). There was a high correlation coefficient (r r =0.972; P 50 =100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl 2 , ZnCl 2 and CuCl 2 inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd 2+ on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15μM. The similarity of the characteristics of the binding of these ligands with that of 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.)

  13. Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

    Science.gov (United States)

    Ozawa, Chihiro; Horiguchi, Michiko; Akita, Tomomi; Oiso, Yuki; Abe, Kaori; Motomura, Tomoki; Yamashita, Chikamasa

    2016-01-01

    Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema.

  14. (3H)leukotriene B4 binding to the guinea pig spleen membranes: a rich tissue source for a high affinity leukotriene B4 receptor site

    International Nuclear Information System (INIS)

    Cheng, J.B.; Kohi, F.; Townley, R.G.

    1986-01-01

    To select a tissue rich for the high affinity leukotriene (LT)B 4 receptor site, they compared binding of 1 nM ( 3 H)LTB 4 (180 Ci/mmol) to the crude membrane preparations of guinea pig spleen, thymus, lung, uterus, bladder, brain, adrenal gland, small intestine, liver, kidney and heart. They found that the membrane preparations from spleen contained the highest binding activity per mg protein. They characterized the LTB 4 binding to the spleen preparation in detail. LTB 4 binding was rapid, reversible, stereoselective and saturable. The data from equilibrium experiments showed a linear Scatchard plot with a K/sub d/ of 1.6 nM and a binding site density of 259 fmol/mg prot. The rank order of agents competing for spleen ( 3 H)LTB 4 binding at 25 0 C was: LTB 4 (K/sub i/ = 2.8 nM) > 20-OH-LTB 4 (23 nM) > LTA 4 (48 nM) > LTA 4 methyl ester (0.13 μM) > 20-COOH-LTB 4 (> 6.6 μM) ≥ arachidonic acid (0.15 mM) similarly ordered FPL-55,712 (0.11 mM). At 4 0 C, LTB 4 (2.3 nM) competed at least 10x more effectively than 20-OH-LTB 4 (29 nM) and 20-COOH-LTB 4 (> 6.6 μM). HPLC analysis indicated that incubation of 84 ng LTB 4 with the spleen membrane at 25 0 C did not result in the formation of 20-OH-LTB 4 ( 3 H)LTB 4 receptor binding sites

  15. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    Science.gov (United States)

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

  16. Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

    Science.gov (United States)

    Braun, Michael B; Traenkle, Bjoern; Koch, Philipp A; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-21

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

  17. High affinity [3H]glibenclamide binding sites in rat neuronal and cardiac tissue: Localization and developmental characteristics

    International Nuclear Information System (INIS)

    Miller, J.A.; Velayo, N.L.; Dage, R.C.; Rampe, D.

    1991-01-01

    We examined the binding of the antidiabetic sulfonylurea [3H] glibenclamide to rat brain and heart membranes. High affinity binding was observed in adult rat forebrain (Kd = 137.3 pM, maximal binding site density = 91.8 fmol/mg of protein) and ventricle (Kd = 77.1 pM, maximal binding site density = 65.1 fmol/mg of protein). Binding site density increased approximately 250% in forebrain membranes during postnatal development but was constant in ventricular membranes. Quantitative autoradiography was used to examine the regional distribution of [3H] glibenclamide binding sites in sections from rat brain, spinal cord and heart. The greatest density of binding in adult brain was found in the substantia nigra and globus pallidus, whereas the other areas displayed heterogenous binding. In agreement with the membrane binding studies, 1-day-old rat brain had significantly fewer [3H]glibenclamide binding sites than adult brain. Additionally, the pattern of distribution of these sites was qualitatively different from that of the adult. In adult rat spinal cord, moderate binding densities were observed in spinal cord gray and displayed a rostral to caudal gradient. In adult rat heart, moderate binding densities were observed and the sites were distributed homogeneously. In conclusion, significant development of [3H]glibenclamide binding sites was seen in the brain but not the heart during postnatal maturation. Furthermore, a heterogeneous distribution of binding sites was observed in both the brain and spinal cord of adult rats

  18. 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.

  19. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

    Science.gov (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

    2018-04-30

    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. 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

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. 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.

  2. High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

    Science.gov (United States)

    Chavan, Tanmay S.; Jang, Hyunbum; Khavrutskii, Lyuba; Abraham, Sherwin J.; Banerjee, Avik; Freed, Benjamin C.; Johannessen, Liv; Tarasov, Sergey G.; Gaponenko, Vadim; Nussinov, Ruth; Tarasova, Nadya I.

    2015-01-01

    Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible. PMID:26682817

  3. 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.

  4. 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.

  5. 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.

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

    International Nuclear Information System (INIS)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M.

    1989-01-01

    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 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

  7. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

    Science.gov (United States)

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

    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 the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  8. 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 leucoc...

  9. 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

  10. 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...

  11. Contrast-enhanced CT with a High-Affinity Cationic Contrast Agent for Imaging ex Vivo Bovine, Intact ex Vivo Rabbit, and in Vivo Rabbit Cartilage

    OpenAIRE

    Stewart, Rachel C.; Bansal, Prashant N.; Entezari, Vahid; Lusic, Hrvoje; Nazarian, Rosalynn M.; Snyder, Brian D.; Grinstaff, Mark W.

    2013-01-01

    The high affinity of a cationic iodinated contrast agent for cartilage provides better tissue visualization, easier segmentation, higher contrast-to-noise ratios, and longer usable imaging windows and requires a lower dose of injected contrast agent compared with an anionic contrast agent.

  12. 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

  13. 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.

  14. High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

    Directory of Open Access Journals (Sweden)

    Stefan Ryser

    Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

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

    Science.gov (United States)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

    2002-07-01

    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 resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal 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 against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed 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, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

  16. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

    Science.gov (United States)

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

    2014-10-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

  17. Antioxidant capacity and contents of phenols, ascorbic acid, β-carotene and lycopene in lettuce

    OpenAIRE

    Zdravković Jasmina M.; Aćamović-Đoković Gordana S.; Mladenović Jelena D.; Pavlović Radoš M.; Zdravković Milan S.

    2014-01-01

    The antioxidant activity of three lettuce varieties (Lactuca sativa L.) Emerald, Vera and Neva, cultivated in two kinds of protected spaces, a glasshouse and a plastic greenhouse, under controlled conditions, was determined. The content of antioxidant compounds: total phenols, flavonoids, L-ascorbic acid, ß-carotene and lycopene, were determined in ethanolic extracts of the lettuce with spectrophotometric methods. The largest content of total phenols (78.98...

  18. Locked nucleic acid

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Sørensen, Mads D; Wengel, Jesper

    2004-01-01

    Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic...

  19. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T. [Samsung Medical Center, Seoul (Korea, Republic of)

    2005-07-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2{alpha} of 5.2 min and late T1/2{beta} of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8{+-}0.5 ml/min, T1/2 of 4.1{+-}0.4 hr, and volume of distribution of 69.7{+-}4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors.

  20. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    International Nuclear Information System (INIS)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T.

    2005-01-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2α of 5.2 min and late T1/2β of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8±0.5 ml/min, T1/2 of 4.1±0.4 hr, and volume of distribution of 69.7±4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors

  1. Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

    Science.gov (United States)

    Birchfield, N B; Casida, J E

    1996-01-01

    Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

  2. Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

    Science.gov (United States)

    Sioud, Mouldy; Westby, Phuong; Vasovic, Vlada; Fløisand, Yngvar; Peng, Qian

    2018-04-16

    mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and

  3. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

    Science.gov (United States)

    Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B.; Larson, Steven M.; Cheung, Nai-Kong

    2014-01-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g. of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive (GD2(+)) tumors. For this purpose, a IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 (1) and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with beta-particle emitting radiometals such as 177Lu and 90Y (2, 3). A three-step regimen including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days (d)) was optimized in immunocompromised mice carrying subcutaneous (s.c.) human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were ∼85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indicies (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5 per group; tumor volume: 240 ± 160 mm3) with three successive PRIT cycles (total 177Lu: ∼33 MBq; tumor dose ∼3400 cGy), revealed complete tumor response in 5/5 animals, with no recurrence up to 28 d post-treatment. Tumor ablation was confirmed histologically in 4/5 mice, and normal organs showed minimal overall toxicities. All non-treated mice required sacrifice within 12 d (>1.0 cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate s.c. GD2(+)–NB in mice while sparing kidney and bone marrow. PMID:24944121

  4. Aortic wall damage in mice unable to synthesize ascorbic acid

    OpenAIRE

    Maeda, Nobuyo; Hagihara, Hiroyuki; Nakata, Yukiko; Hiller, Sylvia; Wilder, Jennifer; Reddick, Robert

    2000-01-01

    By inactivating the gene for l-gulono-γ-lactone oxidase, a key enzyme in ascorbic acid synthesis, we have generated mice that, like humans, depend on dietary vitamin C. Regular chow, containing about 110 mg/kg of vitamin C, is unable to support the growth of the mutant mice, which require l-ascorbic acid supplemented in their drinking water (330 mg/liter). Upon withdrawal of supplementation, plasma and tissue ascorbic acid levels decreased to 10–15% of normal within 2 weeks, and after 5 weeks...

  5. Effects of cytochalasin B on the uptake of ascorbic acid and glucose by 3T3 fibroblasts: Mechanism of impaired ascorbate transport in diabetes

    International Nuclear Information System (INIS)

    Fay, M.J.; Bush, M.J.; Verlangieri, A.J.

    1990-01-01

    Hyperglycemia and/or hypoinsulinemia have been found to inhibit L-ascorbic acid cellular transport. The resultant decrease in intracellular ascorbic acid may de-inhibit aryl sulfatase B and increase degradation of sulfated glycosaminoglycans (sGAG). This could lead to a degeneration of the extracellular matrix and result in increased intimal permeability, the initiating event in atherosclerosis. The present studies show that the glucose transport inhibitor cytochalasin B blocked the uptake of 3 H-2-deoxy-D-glucose by mouse 3T3 fibroblasts. Cytochalasin B also blocked the uptake of 14 C-L-ascorbic acid. The results of these studies further support the hypothesis that glucose and ascorbate share a common transport system. This may have important implications concerning the vascular pathology associated with diabetes mellitus

  6. Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

    International Nuclear Information System (INIS)

    Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

    1989-01-01

    The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

  7. 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...

  8. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    International Nuclear Information System (INIS)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [ 99m Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise

  9. Measurement and COrrelation on Viscosity and Apparent Molar Volume of Ternary System for L—ascorbic Acid in Aqueous D—Glucose and Sucrose Solutions

    Institute of Scientific and Technical Information of China (English)

    赵长伟; 马沛生

    2003-01-01

    Visosities and densities at ,several temperatures from 293.15 K to 313.15K are reported for L-ascorbic acid in aqueous glucose and sucrose solutions at different concentrations.The parameters of density,Viscosity coefficient B and partial molar volume are calculated by regression.The experimental results show that densities and viscositis decrease as temperature increases at the same solute and solvent (glucose and sucrose aueous solution)concentrations,and increase with concentration of glucose and sucrose at the same solute concentration and temperature,B increases with concentration of glucose and sucrose and temaperature,L-ascorbic acid is sturcture-breaker or structure-making for the glucose and sucrose aqueous solutions ,Furthermore,the solute-solvent interactions in ternary systems of water-glucose-electrolyte and water-sucrose-electrolyte are discussed.

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

    Czech Academy of Sciences Publication Activity Database

    Klein, A. B.; Bay, T.; Villumsen, I. S.; Falk-Petersen, C. B.; Marek, Aleš; Frolund, B.; Clausen, R. P.; Hansen, H. D.; Knudsen, G. M.; Wellendorph, P.

    2016-01-01

    Roč. 100, Nov (2016), s. 138-145 ISSN 0197-0186 Institutional support: RVO:61388963 Keywords : quantitative autoradiography * gamma-hydroxybutyric acid * HOCPCA * radioligand * expression * mouse brain development Subject RIV: CE - Biochemistry Impact factor: 3.262, year: 2016

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  12. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August E-mail: schubiger@psi.ch

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [{sup 99m}Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

  13. A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

    Science.gov (United States)

    Fujii, Rika; Schlom, Jeffrey; Hodge, James W

    2018-05-01

    OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for

  14. High-Affinity Methanotrophy Informed by Genome-Wide Analysis of Upland Soil Cluster Alpha (USCα) from Axel Heiberg Island, Canadian High Arctic

    Science.gov (United States)

    Rusley, C.; Onstott, T. C.; Lau, M.

    2017-12-01

    Methane (CH4) is a potent greenhouse gas whose proper budgeting is vital to climate predictions. Recent studies have identified upland Arctic mineral cryosols as consistent CH4 sinks, drawing CH4 from both the atmosphere and underlying anaerobic soil layers. Global atmospheric CH4 uptake is proposed to be mediated by high-affinity methanotrophs based on the detection of the marker gene pmoA (particulate methane monooxygenase beta subunit). However, a lack of pure cultures and scarcity of genomic information have hindered our understanding of their metabolic capabilities and versatility. Together with the missing genetic linkage between its pmoA and 16S ribosomal RNA (rRNA) gene, the factors that control the distribution and magnitude of high-affinity methanotrophy in the Arctic permafrost-affected region have remained elusive. Using 21 metagenomic datasets of surface soils obtained from long-term core incubation experiments,1 this bioinformatics study aimed to reconstruct the draft genome of the Upland Soil Cluster α-proteobacteria (USCα), the high-affinity methanotroph previously detected in the samples,2 and to determine its phylogeny and metabolic requirements. We obtained a genome bin containing the high-affinity form of the USCα-like pmoA gene. The 3.03 Mbp assembly is 91.6% complete with a unique set of single-copy marker genes. The 16S rRNA gene fragment of USCα belongs to the α-proteobacterial family Beijerinckiaceae. Genome annotation indicates possible formaldehyde oxidation via tetrahydromethanopterin-linked C1 transfer pathways, acetate utilization, carbon fixation via the Calvin-Benson-Bassham cycle, and glycogen production. Notably, the key enzymes for formaldehyde assimilation via the serine and ribulose monophosphate pathways are missing. The presence of genes encoding nitrate reductase and hemoglobin suggests adaptation to low O2 under water-logged conditions. Since USCα has versatile carbon metabolisms, it may not be an obligate methanotroph

  15. 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 are ...... of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke....

  16. Z-(-,-)-[76Br]BrQNP: a high affinity PET radiotracer for central and cardiac muscarinic receptors

    International Nuclear Information System (INIS)

    Strijckmans, V.; Coulon, C.; Loc'h, C.; Maziere, B.; Luo, H.; McPherson, D.W.; Knapp, F.F.

    1996-01-01

    Racemic E-1-azabicyclo[2.2.2]oct-3-yl α-(1-bromo-1-1-propen-3-yl)-α -hydroxy-α-phenylacetate (BrQNP) was prepared and evaluated in vivo as a potential candidate for imaging muscarinic acetylcholinergic receptors by Positron Emission Tomography. Initial in vivo blocking studies utilizing Z-(-,-)-[ 125 I]IQNP as a radiolabelled muscarinic probe demonstrated that a preinjection of cold E-BrQNP effectively blocks the uptake of the radiolabelled probe in the brain and heart, by 71% and 86% respectively. Z-(-,-)-[ 76 Br]BrQNP was prepared by electrophilic substitution from a tributylstannyl precursor. Peracetic acid and chloramine T was evaluated as oxidizing agents. After purification by SPE and RP-HPLC, radiolabelling yields of 85% and 95% were obtained with peracetic acid and chloramine T, respectively. The final radiochemical yield was 70% for both oxidizing agents. (author)

  17. 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.

  18. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    Science.gov (United States)

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  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

    International Nuclear Information System (INIS)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H.

    1988-01-01

    Binding studies were performed with two 125 I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-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 δ-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 δ-endotoxins active against M. sexta compete for binding of 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. Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

    Science.gov (United States)

    Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

    2000-08-01

    The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

  1. Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

    Science.gov (United States)

    Hastrup, H; Schwartz, T W

    1996-12-16

    The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

  2. Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Sheppard, P O; Jensen, L B

    2001-01-01

    a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a bacterial periplasmic binding protein. The ATD consists of two globular lobes, which are speculated to contract from an "open" to a "closed" conformation following agonist binding. In the present study, we have...... created a Zn(2+) binding site in mGluR1b by mutating the residue Lys(260) to a histidine. Zinc acts as a noncompetitive antagonist of agonist-induced IP accumulation on the K260H mutant with an IC(50) value of 2 microm. Alanine mutations of three potential "zinc coligands" in proximity to the introduced...

  3. pH, Lactate, and Hypoxia: Reciprocity in Regulating High-Affinity Monocarboxylate Transporter Expression in Glioblastoma

    Directory of Open Access Journals (Sweden)

    James P. Caruso

    2017-02-01

    Full Text Available Highly malignant brain tumors harbor the aberrant propensity for aerobic glycolysis, the excessive conversion of glucose to lactic acid even in the presence of ample tissue oxygen. Lactic acid is rapidly effluxed to the tumor microenvironment via a group of plasma-membrane transporters denoted monocarboxylate transporters (MCTs to prevent “self-poisoning.” One isoform, MCT2, has the highest affinity for lactate and thus should have the ability to respond to microenvironment conditions such as hypoxia, lactate, and pH to help maintain high glycolytic flux in the tumor. Yet, MCT2 is considered to not respond to hypoxia, which is counterintuitive. Its response to tumor lactate has not been reported. In this report, we experimentally identify the transcription initiation site/s for MCT2 in astrocytes (normal and glioma (tumor. We then use a BACmid library to isolate a 4.2-kbp MCT2 promoter-exon I region and examine promoter response to glycolysis-mediated stimuli in glioma cells. Reporter analysis of nested-promoter constructs indicated response of MCT2 to hypoxia, pH, lactate, and glucose, the major physiological “players” that facilitate a tumor's growth and proliferation. Immunoblot analysis of native MCT2 expression under altered pH and hypoxia reflected the reporter data. The pH-mediated gene-regulation studies we describe are the first to record H+-based reporter studies for any mammalian system and demonstrate the exquisite response of the MCT2 gene to minute changes in tumor pH. Identical promoter usage also provides the first evidence of astrocytes harnessing the same gene regulatory regions to facilitate astrocyte-neuron lactate shuttling, a metabolic feature of normal brain.

  4. The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings.

    Science.gov (United States)

    Suchak, Sachin K; Baloyianni, Nicoletta V; Perkinton, Michael S; Williams, Robert J; Meldrum, Brian S; Rattray, Marcus

    2003-02-01

    The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

  5. Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

    Science.gov (United States)

    Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

    1989-07-01

    Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.

  6. Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

    Science.gov (United States)

    Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

    2000-05-01

    CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B

  7. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    International Nuclear Information System (INIS)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-01-01

    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 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 2 Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB 2 Rs (hCB 2 Rs). The affinity of cannabinoids for hCB 2 Rs was determined by competition binding studies employing CHO-hCB 2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB 2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB 2 Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB 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 9 -tetrahydrocannabinol (Δ 9 -THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB 2 R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB 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 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 1 and CB 2 Rs. - Highlights: • JWH-018 and JWH-073 are synthetic cannabinoids present in abused K2

  8. 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

  9. 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.).

  10. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Science.gov (United States)

    Wall, Jonathan S; Williams, Angela; Richey, Tina; Stuckey, Alan; Huang, Ying; Wooliver, Craig; Macy, Sallie; Heidel, Eric; Gupta, Neil; Lee, Angela; Rader, Brianna; Martin, Emily B; Kennel, Stephen J

    2013-01-01

    Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  11. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Directory of Open Access Journals (Sweden)

    Jonathan S Wall

    Full Text Available Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  12. 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

    Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites...... 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......, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in a-amylases....

  13. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    Science.gov (United States)

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

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

    Science.gov (United States)

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

    2013-01-01

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

  15. ADCC employing an NK cell line (haNK) expressing the high affinity CD16 allele with avelumab, an anti-PD-L1 antibody.

    Science.gov (United States)

    Jochems, Caroline; Hodge, James W; Fantini, Massimo; Tsang, Kwong Y; Vandeveer, Amanda J; Gulley, James L; Schlom, Jeffrey

    2017-08-01

    NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In the studies reported here, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Studies also show that while NK cells have the ability to lyse aNK or haNK cells, the addition of NK cells to irradiated haNK cells does not inhibit haNK-mediated lysis of human tumor cells, with or without the addition of avelumab. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells. © 2017 UICC.

  16. Ferulic acid stabilizes a solution of vitamins C and E and doubles its photoprotection of skin.

    Science.gov (United States)

    Lin, Fu-Hsiung; Lin, Jing-Yi; Gupta, Ravindra D; Tournas, Joshua A; Burch, James A; Selim, M Angelica; Monteiro-Riviere, Nancy A; Grichnik, James M; Zielinski, Jan; Pinnell, Sheldon R

    2005-10-01

    Ferulic acid is a potent ubiquitous plant antioxidant. Its incorporation into a topical solution of 15%l-ascorbic acid and 1%alpha-tocopherol improved chemical stability of the vitamins (C+E) and doubled photoprotection to solar-simulated irradiation of skin from 4-fold to approximately 8-fold as measured by both erythema and sunburn cell formation. Inhibition of apoptosis was associated with reduced induction of caspase-3 and caspase-7. This antioxidant formulation efficiently reduced thymine dimer formation. This combination of pure natural low molecular weight antioxidants provides meaningful synergistic protection against oxidative stress in skin and should be useful for protection against photoaging and skin cancer.

  17. An HIV-1 encoded peptide mimics the DNA binding loop of NF-κB and binds thioredoxin with high affinity

    International Nuclear Information System (INIS)

    Su Guoping; Wang Min; Taylor, Ethan Will

    2005-01-01

    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-κB), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-κB 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-κB/Rel family of transcription factors by the putative HIV-1 pro-fs protein

  18. Synthesis and characterization of [{sup 76}Br]-labeled high-affinity A{sub 3} adenosine receptor ligands for positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Kiesewetter, Dale O. [Positron Emission Tomography Radiochemistry Group, NIBIB, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States)], E-mail: dk7k@nih.gov; Lang Lixin; Ma Ying; Bhattacharjee, Abesh Kumar [Positron Emission Tomography Radiochemistry Group, NIBIB, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Gao, Zhan-Guo; Joshi, Bhalchandra V.; Melman, Artem; Castro, Sonia de; Jacobson, Kenneth A. [Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2009-01-15

    Introduction: Bromine-76-radiolabeled analogues of previously reported high-affinity A{sub 3} adenosine receptor (A{sub 3}AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography. Methods: The radiosyntheses were accomplished by oxidative radiobromination on the N{sup 6}-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. Results: We prepared an agonist ligand {l_brace}[{sup 76}Br](1'S,2'R,3'S,4'R,5'S)-4'-{l_brace}2-chloro-6-[(3-bromophenylmethyl)amino] purin-9-yl{r_brace}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581){r_brace} in 59% radiochemical yield with a specific activity of 19.5 GBq/{mu}mol and an antagonist ligand {l_brace}[{sup 76}Br](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino) -2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147){r_brace} in 65% radiochemical yield with a specific activity of 22 GBq/{mu}mol. The resultant products exhibited the expected high affinity (K{sub i}{approx}0.6 nM) and specific binding at the human A{sub 3}AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A{sub 3}AR, over a 2-h time course, which suggests the presence of a specific A{sub 3}AR retention mechanism. Conclusion: We were able to compare uptake of the [{sup 76}Br]-labeled antagonist MRS5147 to [{sup 76}Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A{sub 3}AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.

  19. 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.

  20. 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

    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...... displayed lower affinities (Ki 2.3-347 nM) with highest affinities of GIP(3-30)NH2 and (5-30)NH2 . Agonism was only observed for GIP(1-30)NH2 with an Emax on 100% of GIP(1-42) and GIP(2-30)NH2 (Emax 20%). GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and right-shifts of the GIP(1-42)-response......, 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....

  1. Premature Aging Phenotype in Mice Lacking High-Affinity Nicotinic Receptors: Region-Specific Changes in Layer V Pyramidal Cell Morphology.

    Science.gov (United States)

    Konsolaki, Eleni; Skaliora, Irini

    2015-08-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. Α deficient cholinergic system has been implicated as one of the main factors that could confer a heightened vulnerability to the aging process, and mice lacking high-affinity nicotinic receptors (β2(-/-)) have been proposed as an animal model of accelerated cognitive aging. To date, however, age-related changes in neuronal microanatomy have not been studied in these mice. In the present study, we examine the neuronal structure of yellow fluorescent protein (YFP(+)) layer V neurons in 2 cytoarchitectonically distinct cortical regions in wild-type (WT) and β2(-/-) animals. We find that (1) substantial morphological differences exist between YFP(+) cells of the anterior cingulate cortex (ACC) and primary visual cortex (V1), in both genotypes; (2) in WT animals, ACC cells are more susceptible to aging compared with cells in V1; and (3) β2 deletion is associated with a regionally and temporally specific increase in vulnerability to aging. ACC cells exhibit a prematurely aged phenotype already at 4-6 months, whereas V1 cells are spared in adulthood but strongly affected in old animals. Collectively, our data reveal region-specific synergistic effects of aging and genotype and suggest distinct vulnerabilities in V1 and ACC neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer's disease: an autoradiographic study using 3H-hemicholinium-3

    International Nuclear Information System (INIS)

    Rodriguez-Puertas, R.; Pazos, A.; Zarranz, J.J.; Pascual, J.

    1994-01-01

    H-hemicholinium-3 (H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer's disease (AD) patients and of 11 matched controls. A significant decrease in the density of H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I-III of the frontal cortex. By contrast, in the caudate-putamen the number of H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (-40 %), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD. (author)

  3. High Affinity vs. Native Fibronectin in the Modulation of αvβ3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design.

    Directory of Open Access Journals (Sweden)

    Antonella Paladino

    2017-01-01

    Full Text Available Understanding how binding events modulate functional motions of multidomain proteins is a major issue in chemical biology. We address several aspects of this problem by analyzing the differential dynamics of αvβ3 integrin bound to wild type (wtFN10, agonist or high affinity (hFN10, antagonist mutants of fibronectin. We compare the dynamics of complexes from large-scale domain motions to inter-residue coordinated fluctuations to characterize the distinctive traits of conformational evolution and shed light on the determinants of differential αvβ3 activation induced by different FN sequences. We propose an allosteric model for ligand-based integrin modulation: the conserved integrin binding pocket anchors the ligand, while different residues on the two FN10's act as the drivers that reorganize relevant interaction networks, guiding the shift towards inactive (hFN10-bound or active states (wtFN10-bound. We discuss the implications of results for the design of integrin inhibitors.

  4. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo.

    Science.gov (United States)

    Sankova, Tatiana P; Orlov, Iurii A; Saveliev, Andrey N; Kirilenko, Demid A; Babich, Polina S; Brunkov, Pavel N; Puchkova, Ludmila V

    2017-11-03

    There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell's copper metabolism and its chelating properties are discussed.

  5. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1, Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana P. Sankova

    2017-11-01

    Full Text Available There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST and the N-terminal domain (ectodomain of human high affinity copper transporter CTR1 (hNdCTR1, which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed.

  6. High affinity radiopharmaceuticals based upon lansoprazole for PET imaging of aggregated tau in Alzheimer's disease and progressive supranuclear palsy: synthesis, preclinical evaluation, and lead selection.

    Science.gov (United States)

    Fawaz, Maria V; Brooks, Allen F; Rodnick, Melissa E; Carpenter, Garrett M; Shao, Xia; Desmond, Timothy J; Sherman, Phillip; Quesada, Carole A; Hockley, Brian G; Kilbourn, Michael R; Albin, Roger L; Frey, Kirk A; Scott, Peter J H

    2014-08-20

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [(18)F]lansoprazole, [(11)C]N-methyl lansoprazole, and [(18)F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [(18)F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials.

  7. High Affinity Radiopharmaceuticals Based Upon Lansoprazole for PET Imaging of Aggregated Tau in Alzheimer’s Disease and Progressive Supranuclear Palsy: Synthesis, Preclinical Evaluation, and Lead Selection

    Science.gov (United States)

    2014-01-01

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [18F]lansoprazole, [11C]N-methyl lansoprazole, and [18F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [18F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials. PMID:24896980

  8. Carbon-11 labelling of eticlopride in two different positions - a selective high-affinity ligand for the study of dopamine D-2 receptors using PET

    International Nuclear Information System (INIS)

    Halldin, Christer; Hall, Haakan

    1990-01-01

    A new highly selective high-affinity dopamine D-2 receptor antagonist, eticlopride ((-)-(S)-5-chloro-3-ethyl-N-(1-ethyl-2-pyrrolidinyl)methyl)-6-methoxysalicylamide), was labelled with 11 C in two different positions ([N-ethyl- 11 C]eticlopride (I) and ([methyl- 11 C]eticlopride (II)). Product I was prepared by N-alkylation of the N-desethyl compound with [ 11 C]ethyl iodide. II was prepared by O-alkylation of the diphenolic precursor with [ 11 C]methyl iodide followed by separation of the two methylated products. The radiochemical yields were 15-20% (EOB) with an overall synthesis time of 45-60 min. Both compounds were isolated by semi-preparative HPLC and the radiochemical purity was in both cases > 99%. I was injected i.v. in a Cynomolgus monkey and brain radioactivity was measured by positron emission tomography (PET). The specific activity was 70 Ci/mmol at time of injection. There was a marked accumulation of radioactivity in the basal ganglia, regions known to have a high density of dopamine D-2 receptors. (author)

  9. Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

    Science.gov (United States)

    Miyake, Tsuyoshi; Hiraishi, Hiroyuki; Sammoto, Hiroyuki; Ono, Bun-Ichiro

    2003-10-10

    The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

  10. Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer`s disease: an autoradiographic study using {sup 3}H-hemicholinium-3

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Puertas, R; Pazos, A [Dept. of Physiology and Pharmacology, Unit of Pharmacology, Univ. of Cantabria, Santander (Spain); Zarranz, J J [Dept. of Neuroscience, Univ. of the Basque Country, Leioa (Spain); Pascual, J [Dept. of Medicine, Service of Neurology, Univ. Hospital ` Marques de Valdecilla` , Univ. of Cantabria, Santander (Spain)

    1994-12-31

    H-hemicholinium-3 (H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer`s disease (AD) patients and of 11 matched controls. A significant decrease in the density of H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I-III of the frontal cortex. By contrast, in the caudate-putamen the number of H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (-40 %), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD. (author).

  11. PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family.

    Science.gov (United States)

    Quesada, A; Krapp, A; Trueman, L J; Daniel-Vedele, F; Fernández, E; Forde, B G; Caboche, M

    1997-05-01

    A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homologues of this family have been cloned from a higher plant (barley). Based on six of the peptide sequences most strongly conserved between the barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from other plant species. The utility of these primers was demonstrated by RT-PCR with cDNA made from poly(A)+ RNA from barley, C. reinhardtii and Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbaginifolia was used as probe to isolate a full-length cDNA clone which encodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumbaginifolia. Northern blotting showed that the Nrt2 transcripts are most strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reductase genes: their expression was strongly induced by nitrate but was repressed when reduced forms of N were supplied to the roots.

  12. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo

    Directory of Open Access Journals (Sweden)

    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. 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.

  14. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and σligands to guinea pig brain

    International Nuclear Information System (INIS)

    Klein, M.; Canoll, P.D.; Musacchio, J.M.

    1991-01-01

    The DM 1 /σ 1 site binds dextromethorphan (DM) and σ 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 [ 3 H]dextromethorphan, [ 3 H]3-(3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[ 3 H]1,3-Di-o-tolyl-guanidine ([ 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 i values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM 1 σ 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K i values of 9-13 and 3-4 μM respectively against the three labeled ligands. These results, the broad specificity of the DM 1 /σ 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

  15. [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.

  16. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors

    International Nuclear Information System (INIS)

    Amagai, Yosuke; Tanaka, Akane; Ohmori, Keitaro; Matsuda, Hiroshi

    2008-01-01

    Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (FcεRI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with FcεRI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders

  17. Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

    NARCIS (Netherlands)

    de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

    1994-01-01

    The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a

  18. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-10-01

    Full Text Available Abstract Background For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND, the potent drug for treatment of multiple myeloma (MM, a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. Results In this study, a hapten of LND (N-glutaryl-LND was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA and keyhole limpet hemocyanin (KLH proteins by ethyl-3-(3-dimethylaminopropyl carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. Conclusions The high affinity of the antibody (IC50 = 10 ng/mL will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  19. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma.

    Science.gov (United States)

    Darwish, Ibrahim A; Alzoman, Nourh Z; Abuhejail, Reem M; El-Samani, Tilal E

    2012-10-26

    For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. In this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. The high affinity of the antibody (IC50 = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  20. Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

    Science.gov (United States)

    Chen, Jiandong

    2016-01-01

    ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression. PMID:27849174

  1. Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

    Science.gov (United States)

    Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

    2018-01-01

    Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

  2. High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle

    International Nuclear Information System (INIS)

    Ho, S.M.; Fehrer, S.; Yu, M.; Liang, L.C.; Press, D.

    1988-01-01

    Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species

  3. The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis.

    Science.gov (United States)

    Qi, Zhi; Hampton, Corrina R; Shin, Ryoung; Barkla, Bronwyn J; White, Philip J; Schachtman, Daniel P

    2008-01-01

    Caesium (Cs(+)) is a potentially toxic mineral element that is released into the environment and taken up by plants. Although Cs(+) is chemically similar to potassium (K(+)), and much is known about K(+) transport mechanisms, it is not clear through which K(+) transport mechanisms Cs(+) is taken up by plant roots. In this study, the role of AtHAK5 in high affinity K(+) and Cs(+) uptake was characterized. It is demonstrated that AtHAK5 is localized to the plasma membrane under conditions of K(+) deprivation, when it is expressed. Growth analysis showed that AtHAK5 plays a role during severe K(+) deprivation. Under K(+)-deficient conditions in the presence of Cs(+), Arabidopsis seedlings lacking AtHAK5 had increased inhibition of root growth and lower Cs(+) accumulation, and significantly higher leaf chlorophyll concentrations than wild type. These data indicate that, in addition to transporting K(+) in planta, AtHAK5 also transports Cs(+). Further experiments showed that AtHAK5 mediated Cs(+) uptake into yeast cells and that, although the K(+) deficiency-induced expression of AtHAK5 was inhibited by low concentrations of NH(4)(+) in planta, Cs(+) uptake by yeast was stimulated by low concentrations of NH(4)(+). Interestingly, the growth of the Arabidopsis atakt1-1 mutant was more sensitive to Cs(+) than the wild type. This may be explained, in part, by increased expression of AtHAK5 in the atakt1-1 mutant. It is concluded that AtHAK5 is a root plasma membrane uptake mechanism for K(+) and Cs(+) under conditions of low K(+) availability.

  4. Preliminary assessment of extrastriatal dopamine d-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, {sup 18}F-fallypride

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Jogeshwar E-mail: jogeshwar-mukherjee@ketthealth.com; Yang, Z.-Y.; Brown, Terry; Lew, Robert; Wernick, Miles; Ouyang Xiaohu; Yasillo, Nicholas; Chen, C.-T.; Mintzer, Robert; Cooper, Malcolm

    1999-07-01

    We have identified the value of {sup 18}F-fallypride {l_brace}(S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[{sup 18}F]fluoropropyl)-2,3-dim= ethoxybenzamide{r_brace}, as a dopamine D-2 receptor radiotracer for the study of striatal and extrastriatal receptors. Fallypride exhibits high affinities for D-2 and D-3 subtypes and low affinity for D-4 ({sup 3}H-spiperone IC{sub 50}s: D-2=0.05 nM [rat striata], D-3=0.30 nM [SF9 cell lines, rat recombinant], and D-4=240 nM [CHO cell lines, human recombinant]). Biodistribution in the rat brain showed localization of {sup 18}F-fallypride in striata and extrastriatal regions such as the frontal cortex, parietal cortex, amygdala, hippocampus, thalamus, and hypothalamus. In vitro autoradiographic studies in sagittal slices of the rat brain showed localization of {sup 18}F-fallypride in striatal and several extrastriatal regions, including the medulla. Positron emission tomography (PET) experiments with {sup 18}F-fallypride in male rhesus monkeys were carried out in a PET VI scanner. In several PET experiments, apart from the specific binding seen in the striatum, specific binding of {sup 18}F-fallypride was also identified in extracellular regions (in a lower brain slice, possibly the thalamus). Specific binding in the extrastriata was, however, significantly lower compared with that observed in the striata of the monkeys (extrastriata/cerebellum = 2, striata/cerebellum = 10). Postmortem analysis of the monkey brain revealed significant {sup 18}F-fallypride binding in the striata, whereas binding was also observed in extrastriatal regions such as the thalamus, cortical areas, and brain stem.

  5. SNF3 as high affinity glucose sensor and its function in supporting the viability of Candida glabrata under glucose-limited environment

    Directory of Open Access Journals (Sweden)

    Tzu Shan eNg

    2015-12-01

    Full Text Available Candida glabrata is an emerging human fungal pathogen that has efficacious nutrient sensing and responsiveness ability. It can be seen through its ability to thrive in diverse range of nutrient limited-human anatomical sites. Therefore, nutrient sensing particularly glucose sensing is thought to be crucial in contributing to the development and fitness of the pathogen. This study aimed to elucidate the role of SNF3 (Sucrose Non Fermenting 3 as a glucose sensor and its possible role in contributing to the fitness and survivability of C. glabrata in glucose-limited environment. The SNF3 knockout strain was constructed and subjected to different glucose concentrations to evaluate its growth, biofilm formation, amphotericin B susceptibility, ex vivo survivability and effects on the transcriptional profiling of the sugar receptor repressor (SRR pathway-related genes. The SNF3Δ strain showed a retarded growth in low glucose environments (0.01% and 0.1% in both fermentation and respiration-preferred conditions but grew well in high glucose concentration environments (1% and 2%. It was also found to be more susceptible to amphotericin B in low glucose environment (0.1% and macrophage engulfment but showed no difference in the biofilm formation capability. The deletion of SNF3 also resulted in the down-regulation of about half of hexose transporters genes (4 out of 9. Overall, the deletion of SNF3 causes significant reduction in the ability of C. glabrata to sense limited surrounding glucose and consequently disrupts its competency to transport and perform the uptake of this critical nutrient. This study highlighted the role of SNF3 as a high affinity glucose sensor and its role in aiding the survivability of C. glabrata particularly in glucose limited environment.

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

    Science.gov (United States)

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

    2016-04-01

    The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3(-)), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To explore the effects and mode of action of NO3(-) on Lpr, we used an extended set of NO3(-) transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1 and nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3(-) conditions. First, a strong positive relationship between Lpr and NO3(-) accumulation, in shoots rather than in roots, was revealed. Secondly, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role for the high-affinity NO3(-) transporter NRT2.1 in increasing Lpr These results indicate that NO3(-)signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative real-time reverse transcription-PCR and enzyme-linked immunosorbent assays (ELISAs) were used to investigate the effects of NO3(-) availability on plasma membrane aquaporin (plasma membrane intrinsic protein; PIP) expression. Whereas PIP regulation mostly occurs at the post-translational level in wild-type plants, a regulation of PIPs at both the transcriptional and translational levels was uncovered in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3(-) involves novel shoot to root signaling and NRT2.1-dependent functions. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines

    Directory of Open Access Journals (Sweden)

    Lea Hiršl

    2018-05-01

    Full Text Available The development of a vaccine against human cytomegalovirus (CMV has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1 (MULT-1MCMV inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector.

  8. High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71.

    Science.gov (United States)

    Levengood, Jeffrey D; Tolbert, Michele; Li, Mei-Ling; Tolbert, Blanton S

    2013-07-01

    Human Enterovirus 71 (EV71) is an emerging pathogen of infectious disease and a serious threat to public health. Currently, there are no antivirals or vaccines to slow down or prevent EV71 infections, thus underscoring the urgency to better understand mechanisms of host-enterovirus interactions. EV71 uses a type I internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit via a pathway that requires the cytoplasmic localization of hnRNP A1, which acts as an IRES trans-activating factor. The mechanism of how hnRNP A1 trans activates EV71 RNA translation is unknown, however. Here, we report that the UP1 domain of hnRNP A1 interacts specifically with stem loop II (SLII) of the IRES, via a thermodynamically well-defined biphasic transition that involves conserved bulge 5'-AYAGY-3' and hairpin 5'-RY(U/A)CCA-3' loops. Calorimetric titrations of wild-type and mutant SLII constructs reveal these structural elements are essential to form a high-affinity UP1-SLII complex. Mutations that alter the bulge and hairpin primary or secondary structures abrogate the biphasic transition and destabilize the complex. Notably, mutations within the bulge that destabilize the complex correlate with a large reduction in IRES-dependent translational activity and impair EV71 replication. Taken together, this study shows that a conserved SLII structure is necessary to form a functional hnRNP A1-IRES complex, suggesting that small molecules that target this stem loop may have novel antiviral properties.

  9. Further Studies on Oxalic Acid Biosynthesis in Oxalate-accumulating Plants 1

    Science.gov (United States)

    Nuss, Richard F.; Loewus, Frank A.

    1978-01-01

    l-Ascorbic acid functions as a precursor of oxalic acid in several oxalate-accumulating plants. The present study extends this observation to include Rumex crispus L. (curly dock), Amaranthus retroflexus L. (red root pigweed), Chenopodium album L. (lamb's-quarters), Beta vulgaris L. (sugar beet), Halogeton glomeratus M. Bieb. (halogeton), and Rheum rhabarbarum L. (rhubarb). Several species with low oxalate content are also examined. When l-[1-14C]ascorbic acid is supplied to young seedlings of R. crispus or H. glomeratus, a major portion of the 14C is released over a 24-hour period as 14CO2 and only a small portion is recovered as [14C]oxalate, unlike cuttings from 2- or 4-month-old plants which retain a large part of the 14C as [14C]oxalic acid and release very little 14CO2. Support for an intermediate role of oxalate in the release of 14CO2 from l-[1-14C]ascorbic acid is seen in the rapid release of 14CO2 by R. crispus and H. glomeratus seedlings labeled with [14C]oxalic acid. The common origin of oxalic acid carbon in the C1 and C2 fragment from l-ascorbic acid is demonstrated by comparison of 14C content of oxalic acid in several oxalate-accumulators after cuttings or seedlings are supplied equal amounts of l-[1-14C]- or l-[UL-14C]ascorbic acid. Theoretically, l-[1-14C]ascorbic acid will produce labeled oxalic acid containing three times as much 14C as l-[UL-14C]ascorbic acid when equal amounts of label are provided. Experimentally, a ratio of 2.7 ± 0.5 is obtained in duplicate experiments with six different species. PMID:16660342

  10. PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    2010-05-01

    Full Text Available Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases and hydrolysis (by cAMP phosphodiesterases. We functionally characterized gene-deletion mutants of a high-affinity (PdeH and a low-affinity (PdeL cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold, precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling

  11. 2,2'-Dithiobis(N-ethyl-spermine-5-carboxamide) is a high affinity, membrane-impermeant antagonist of the mammalian polyamine transport system.

    Science.gov (United States)

    Huber, M; Pelletier, J G; Torossian, K; Dionne, P; Gamache, I; Charest-Gaudreault, R; Audette, M; Poulin, R

    1996-11-01

    We have synthesized 2,2'-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian cells. DESC was the most potent antagonist of spermine transport in ZR-75-1 human breast cancer cells, with Ki values of 5. 0 +/- 0.7, 80 +/- 31, and 16 +/- 3 microM for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cells (Ki = 1.6 +/- 0.5 and 2.7 +/- 1.1 microM, respectively). While DESC and MESC were purely competitive inhibitors of putrescine transport, DEASC was a mixed competitive/noncompetitive antagonist. Remarkably, DESC was virtually impermeant in ZR-75-1 cells despite its low Ki toward polyamine transport. The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polyamine transporter might simultaneously interact with more than one binding site. While DESC strongly decreased the initial rate of [3H]spermidine transport, even a 40-fold molar excess of antagonist could not completely abolish intracellular spermidine accumulation. Moreover, as little as 0.3 microM spermidine fully restored growth in ZR-75-1 cells treated with an inhibitor of polyamine biosynthesis in the presence of 50 microM DESC, thus emphasizing the importance of uptake of trace amounts of exogenous polyamines. Thus, reducing the exogenous supply of polyamines with a potent competitive inhibitor may be kinetically inadequate to block replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demonstrate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provide a framework for the design of

  12. Ascorbic acid and melatonin reduce heat-induced performance inhibition and oxidative stress in Japanese quails.

    Science.gov (United States)

    Sahin, N; Onderci, M; Sahin, K; Gursu, M F; Smith, M O

    2004-02-01

    1. The effects of ascorbic acid (L-ascorbic acid) and melatonin supplementation on performance, carcase characteristics, malondialdehyde (MDA) as lipid peroxidation indicator, ascorbic acid, retinol, tocopherol and mineral status in the Japanese quail (Coturnix coturnix japonica) exposed to high ambient temperature were evaluated. 2. Two hundred and forty Japanese quails (10 d old) were randomly assigned to 8 treatment groups consisting of 10 replicates of three birds each. The birds were kept in a temperature-controlled room at 22 degrees C (Thermoneutral, TN groups) or 34 degrees C (for 8 h/d; 09:00 to 17:00 h; Heat stress, HS groups). Birds in both TN and HS were fed either a basal (control) diet or the basal diet supplemented with 250 mg of L-ascorbic acid/kg of diet (Ascorbic acid group), 40 mg of melatonin/kg of diet (Melatonin group) or both (Ascorbic acid + Melatonin group). 3. Supplementing heat-stressed quails with ascorbic acid and melatonin improved performance compared with the control group. Effects generally were greatest in quails supplemented with both ascorbic acid and melatonin. 4. Although supplementation did not consistently restore the concentrations of serum ascorbic acid, retinol and tocopherol to those of TN groups, these concentrations increased significantly with supplementation. Furthermore, serum and liver MDA and serum cholesterol and glucose concentrations were lower in the supplemented groups than in the heat-stressed controls. 5. Within each environment, excretion of Ca, P, Mg, Zn, Fe and Cr were lowest in the combination group and, in all cases, highest in the HS group. Interactions between diet and temperature were detected for live weight gain, cold carcase weight, MDA, ascorbic acid, tocopherol concentrations and excretion of zinc. 6. The results of the study indicate that ascorbic acid and melatonin supplementation attenuate the decline in performance and antioxidant and mineral status caused by heat stress and such

  13. Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    Energy Technology Data Exchange (ETDEWEB)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C. (National Institute of Mental Health, Bethesda, MD (USA))

    1991-02-01

    Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.

  14. Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    International Nuclear Information System (INIS)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C.

    1991-01-01

    Equilibrium binding studies with the sigma receptor ligand [ 3 H]1,3-di(2-tolyl)guanidine ([ 3 H]DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. [Life Sci. 45:1721-1732 (1989)]. Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that [ 3 H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [ 3 H]DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of [ 3 H]DTG from site 2, suggesting an association of this binding site with calcium channels

  15. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    International Nuclear Information System (INIS)

    Leff, S.E.; Creese, I.

    1985-01-01

    The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

  16. Antioxidant capacity and contents of phenols, ascorbic acid, β-carotene and lycopene in lettuce

    Directory of Open Access Journals (Sweden)

    Zdravković Jasmina M.

    2014-01-01

    Full Text Available The antioxidant activity of three lettuce varieties (Lactuca sativa L. Emerald, Vera and Neva, cultivated in two kinds of protected spaces, a glasshouse and a plastic greenhouse, under controlled conditions, was determined. The content of antioxidant compounds: total phenols, flavonoids, L-ascorbic acid, ß-carotene and lycopene, were determined in ethanolic extracts of the lettuce with spectrophotometric methods. The largest content of total phenols (78.98 ± 0.67 mg GAE/g of dry extract was found in ethanolic extract of the lettuce variety Neva cultivated in a plastic greenhouse, whereas the largest content of flavonoids (35.45 ± 0.95 mg RU/g of dry extract was displayed in the lettuce Emerald cultivated in a glasshouse. It was observed that the lettuce cultivated in the glasshouse contained a somewhat higher content of L-ascorbic acid than the lettuce same variety from plastic greenhouse. The content of lycopene in the examined lettuce is negligible, and the content of ß-carotene is low. On the other hand, the high content of phenolic components causes favourable antioxidant properties found in all varieties of examined lettuce. [Projekat Ministarstva nauke Republike Srbije, br. TR 31059: A new concept in breeding vegetable cultivars and hybrids designed for sustainable growing systems using biotechnological methods

  17. Post-transcriptional regulation of the arginine transporter Cat-1 by amino acid availability

    NARCIS (Netherlands)

    Aulak, K. S.; Mishra, R.; Zhou, L.; Hyatt, S. L.; de Jonge, W.; Lamers, W.; Snider, M.; Hatzoglou, M.

    1999-01-01

    The regulation of the high affinity cationic amino acid transporter (Cat-1) by amino acid availability has been studied. In C6 glioma and NRK kidney cells, cat-1 mRNA levels increased 3.8-18-fold following 2 h of amino acid starvation. The transcription rate of the cat-1 gene remained unchanged

  18. Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex

    Energy Technology Data Exchange (ETDEWEB)

    Marcella, Aaron M.; Culbertson, Sannie J.; Shogren-Knaak, Michael A.; Barb, Adam W.

    2017-11-01

    The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05 and 4.10 Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD = 62 ± 13 nM, followed by the binding of two more equivalents of holo-ACPP with KD = 1.2 ± 0.2 μM. Cooperativity was not observed for apo-ACPP which bound with KD = 2.4 ± 0.1 μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.

  19. Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex.

    Science.gov (United States)

    Marcella, Aaron M; Culbertson, Sannie J; Shogren-Knaak, Michael A; Barb, Adam W

    2017-11-24

    The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a K D =62±13nM, followed by the binding of two more equivalents of holo-ACPP with K D =1.2±0.2μM. Cooperativity was not observed for apo-ACPP which bound with K D =2.4±0.1μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

    Science.gov (United States)

    Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

    1992-01-01

    Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

  1. DOTA-NOC, a high-affinity ligand of somatostatin receptor subtypes 2, 3 and 5 for labelling with various radiometals

    International Nuclear Information System (INIS)

    Wild, Damian; Schmitt, Joerg S.; Ginj, Mihaela; Maecke, Helmut R.; Bernard, Bert F.; Krenning, Eric; Jong, Marion de; Wenger, Sandra; Reubi, Jean-Claude

    2003-01-01

    Earlier studies have shown that modification of the octapeptide octreotide in positions 3 and 8 may result in compounds with increased somatostatin receptor affinity that, if radiolabelled, display improved uptake in somatostatin receptor-positive tumours. The aim of a recent research study in our laboratory was to employ the parallel peptide synthesis approach by further exchanging the amino acid in position 3 of octreotide and coupling the macrocyclic chelator DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to these peptides for labelling with radiometals like gallium-67 or -68, indium-111, yttrium-90 and lutetium-177. The purpose was to find radiopeptides with an improved somatostatin receptor binding profile in order to extend the spectrum of targeted tumours. A first peptide, [ 111 In, 90 Y-DOTA]-1-Nal 3 -octreotide ( 111 In, 90 Y-DOTA-NOC), was isolated which showed an improved profile. In III -DOTA-NOC exhibited the following IC 50 values (nM) when studied in competition with [ 125 I][Leu 8 , d-Trp 22 , Tyr 25 ]somatostatin-28 (values for Y III -DOTA-NOC are shown in parentheses): sstr2, 2.9±0.1 (3.3±0.2); sstr3, 8±2 (26±1.9); sstr5, 11.2±3.5 (10.4±1.6). Affinity towards sstr1 and 4 was very low or absent. In III -DOTA-NOC is superior to all somatostatin-based radiopeptides having this particular type of binding profile, including DOTA-lanreotide, and has three to four times higher binding affinity to sstr2 than In III ,Y III -DOTA-Tyr 3 -octreotide (In III ,Y III -DOTA-TOC). In addition, [ 111 In]DOTA-NOC showed a specific and high rate of internalization into AR4-2J rat pancreatic tumour cells which, after 4 h, was about two times higher than that of [ 111 In]DOTA-TOC and three times higher than that of [ 111 In]DOTA-octreotide ([ 111 In]DOTA-OC). The internalized radiopeptides were externalized intact upon 2 h of internalization followed by an acid wash. After 2-3 h of externalization a plateau is reached, indicating a steady

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

    Science.gov (United States)

    Lu, Shuo; Zgurskaya, Helen I

    2013-11-01

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

  3. SCM, a novel M-like protein from Streptococcus canis, binds (mini)-plasminogen with high affinity and facilitates bacterial transmigration.

    Science.gov (United States)

    Fulde, Marcus; Rohde, Manfred; Hitzmann, Angela; Preissner, Klaus T; Nitsche-Schmitz, D Patric; Nerlich, Andreas; Chhatwal, Gursharan Singh; Bergmann, Simone

    2011-03-15

    Streptococcus canis is an important zoonotic pathogen capable of causing serious invasive diseases in domestic animals and humans. In the present paper we report the binding of human plasminogen to S. canis and the recruitment of proteolytically active plasmin on its surface. The binding receptor for plasminogen was identified as a novel M-like protein designated SCM (S. canis M-like protein). SPR (surface plasmon resonance) analyses, radioactive dot-blot analyses and heterologous expression on the surface of Streptococcus gordonii confirmed the plasminogen-binding capability of SCM. The binding domain was located within the N-terminus of SCM, which specifically bound to the C-terminal part of plasminogen (mini-plasminogen) comprising kringle domain 5 and the catalytic domain. In the presence of urokinase, SCM mediated plasminogen activation on the bacterial surface that was inhibited by serine protease inhibitors and lysine amino acid analogues. Surface-bound plasmin effectively degraded purified fibrinogen as well as fibrin clots, resulting in the dissolution of fibrin thrombi. Electron microscopic illustration and time-lapse imaging demonstrated bacterial transmigration through fibrinous thrombi. The present study has led, for the first time, to the identification of SCM as a novel receptor for (mini)-plasminogen mediating the fibrinolytic activity of S. canis.

  4. Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

    Science.gov (United States)

    Lorkin, P A; Stephens, A D; Beard, M E; Wrigley, P F; Adams, L; Lehmann, H

    1975-01-01

    A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident. PMID:124

  5. Ab initio and DFT study of hydrogen bond interactions between ascorbic acid and dimethylsulfoxide based on FT-IR and FT-Raman spectra

    Science.gov (United States)

    Niazazari, Naser; Zatikyan, Ashkhen L.; Markarian, Shiraz A.

    2013-06-01

    The hydrogen bonding of 1:1 complexes formed between L-ascorbic acid (LAA) and dimethylsulfoxide (DMSO) has been studied by means of ab initio and density functional theory (DFT) calculations. Solutions of L-ascorbic acid (AA) in dimethylsulfoxide (DMSO) have been studied by means of both FT-IR (4000-220 cm-1) and FT-Raman spectroscopy. Ab initio Hartree-Fock (HF) and DFT methods have been used to determine the structure and energies of stable conformers of various types of L-AA/DMSO complexes in gas phase and solution. The basis sets 6-31++G∗∗ and 6-311+G∗ were used to describe the structure, energy, charges and vibrational frequencies of interacting complexes in the gas phase. The optimized geometric parameters and interaction energies for various complexes at different theories have been estimated. Binding energies have been corrected for basis set superposition error (BSSE) and harmonic vibrational frequencies of the structures have been calculated to obtain the stable forms of the complexes. The self-consistent reaction field (SCRF) has been used to calculate the effect of DMSO as the solvent on the geometry, energy and charges of complexes. The solvent effect has been studied using the Onsager models. It is shown that the polarity of the solvent plays an important role on the structures and relative stabilities of different complexes. The results obtained show that there is a satisfactory correlation between experimental and theoretical predictions.

  6. Synthesis, in vitro validation and in vivo pharmacokinetics of [{sup 125}I]N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl) ethylamine: A high-affinity ligand for imaging sigma receptor positive tumors

    Energy Technology Data Exchange (ETDEWEB)

    John, Christy S; Gulden, Mary E; Vilner, Bertold J; Bowen, Wayne D

    1996-08-01

    N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl)ethylamine, IPEMP, and the corresponding bromo derivative, BrPEMP, have been synthesized and characterized. Both BrPEMP and IPEMP were evaluated for sigma-1 and sigma-2 subtype receptor affinities and found to possess very high affinities for both receptor subtypes. The precursor for radioiodination n-tributylstannylphenylethylpiperidinylethylamine was prepared from its bromo derivative by palladium-catalyzed stannylation reaction. Radioiodinated 4-[{sup 125}I]PEMP was readily prepared in high yields and high specific activity by oxidative iododestannylation reaction using chloramine-T as oxidizing agent. Sites labeled by 4-[{sup 125}I]PEMP in guinea pig brain membranes showed high affinity for BD1008, haloperidol, and (+)-pentazocine (Ki = 5.06 {+-} 0.40, 32.6 {+-} 2.75, and 48.1 {+-} 8.60 nM, respectively), which is consistent with sigma receptor pharmacology. Competition binding studies of 4-[{sup 125}I]PEMP in melanoma (A375) and MCF-7 breast cancer cells showed a high affinity, dose-dependent inhibition of binding with known sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl) ethylamine, BD1008 (Ki = 5, 11 nM, respectively), supporting the labeling of sigma sites in these cells. Haloperidol, however showed a weaker (Ki 100-200 nM) affinity for the sites labeled by 4-[{sup 125}I]PEMP in these cells. Biodistribution studies of 4-[{sup 125}I]PEMP in rats showed a fast clearance of this radiopharmaceutical from blood, liver, lung, and other organs. A co-injection of 4-IPEMP with 4-[{sup 125}I]PEMP resulted in 37%, 69%, and 35% decrease in activity in liver, kidney, and brain (organs possessing sigma receptors), respectively at 1-h postinjection. These results suggest that 4-[{sup 125}I]PEMP is a promising radiopharmaceutical for pursuing further studies in animal models with tumors.

  7. A shortcut to high-affinity Ga-68 and Cu-64 radiopharmaceuticals: one-pot click chemistry trimerisation on the TRAP platform.

    Science.gov (United States)

    Baranyai, Zsolt; Reich, Dominik; Vágner, Adrienn; Weineisen, Martina; Tóth, Imre; Wester, Hans-Jürgen; Notni, Johannes

    2015-06-28

    Due to its 3 carbonic acid groups being available for bioconjugation, the TRAP chelator (1,4,7-triazacyclononane-1,4,7-tris(methylene(2-carboxyethylphosphinic acid))) is chosen for the synthesis of trimeric bioconjugates for radiolabelling. We optimized a protocol for bio-orthogonal TRAP conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), including a detailed investigation of kinetic properties of Cu(II)-TRAP complexes. TRAP building blocks for CuAAC, TRAP(alkyne)3 and TRAP(azide)3 were obtained by amide coupling of propargylamine/3-azidopropyl-1-amine, respectively. For Cu(II) complexes of neat and triply amide-functionalized TRAP, the equilibrium properties as well as pseudo-first-order Cu(II)-transchelation, using 10 to 30 eq. of NOTA and EDTA, were studied by UV-spectrophotometry. Dissociation of any Cu(II)-TRAP species was found to be independent on the nature or excess of a competing chelator, confirming a proton-driven two-step mechanism. The respective thermodynamic stability constants (log K(ML): 19.1 and 17.6) and dissociation rates (k: 38 × 10(-6) and 7 × 10(-6) s(-1), 298 K, pH 4) show that the Cu(II) complex of the TRAP-conjugate possesses lower thermodynamic stability but higher kinetic inertness. At pH 2-3, its demetallation with NOTA was complete within several hours/days at room temperature, respectively, enabling facile Cu(II) removal after click coupling by direct addition of NOTA trihydrochloride to the CuAAC reaction mixture. Notwithstanding this, an extrapolated dissociation half life of >100 h at 37 °C and pH 7 confirms the suitability of TRAP-bioconjugates for application in Cu-64 PET (cf. t(1/2)(Cu-64) = 12.7 h). To showcase advantages of the method, TRAP(DUPA-Pep)3, a trimer of the PSMA inhibitor DUPA-Pep, was synthesized using 1 eq. TRAP(alkyne)3, 3.3 eq. DUPA-Pep-azide, 10 eq. Na ascorbate, and 1.2 eq. Cu(II)-acetate. Its PSMA affinity (IC50), determined by the competition assay on LNCa

  8. A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom.

    Science.gov (United States)

    Lebrun, B; Romi-Lebrun, R; Martin-Eauclaire, M F; Yasuda, A; Ishiguro, M; Oyama, Y; Pongs, O; Nakajima, T

    1997-11-15

    A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).

  9. In vitro and in vivo evaluation of a (18F-labeled high affinity NOTA conjugated bombesin antagonist as a PET ligand for GRPR-targeted tumor imaging.

    Directory of Open Access Journals (Sweden)

    Zohreh Varasteh

    Full Text Available Expression of the gastrin-releasing peptide receptor (GRPR in prostate cancer suggests that this receptor can be used as a potential molecular target to visualize and treat these tumors. We have previously investigated an antagonist analog of bombesin (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA via a diethylene glycol (PEG2 spacer (NOTA-P2-RM26 labeled with (68Ga and (111In. We found that this conjugate has favorable properties for in vivo imaging of GRPR-expression. The focus of this study was to develop a (18F-labelled PET agent to visualize GRPR. NOTA-P2-RM26 was labeled with (18F using aluminum-fluoride chelation. Stability, in vitro binding specificity and cellular processing tests were performed. The inhibition efficiency (IC50 of the [(natF]AlF-NOTA-P2-RM26 was compared to that of the (natGa-loaded peptide using (125I-Tyr(4-BBN as the displacement radioligand. The pharmacokinetics and in vivo binding specificity of the compound were studied. NOTA-P2-RM26 was labeled with (18F within 1 h (60-65% decay corrected radiochemical yield, 55 GBq/µmol. The radiopeptide was stable in murine serum and showed high specific binding to PC-3 cells. [(natF]AlF-NOTA-P2-RM26 showed a low nanomolar inhibition efficiency (IC50=4.4±0.8 nM. The internalization rate of the tracer was low. Less than 14% of the cell-bound radioactivity was internalized after 4 h. The biodistribution of [(18F]AlF-NOTA-P2-RM26 demonstrated rapid blood clearance, low liver uptake and low kidney retention. The tumor uptake at 3 h p.i. was 5.5±0.7 %ID/g, and the tumor-to-blood, -muscle and -bone ratios were 87±42, 159±47, 38±16, respectively. The uptake in tumors, pancreas and other GRPR-expressing organs was significantly reduced when excess amount of non-labeled peptide was co-injected. The low uptake in bone suggests a high in vivo stability of the Al-F bond. High contrast PET image was obtained 3 h p

  10. Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    International Nuclear Information System (INIS)

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-01-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [ 3 H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [ 3 H]flunitrazepam and [ 3 H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [ 3 H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy

  11. Role of sialic acid in synaptosomal transport of amino acid transmitters

    International Nuclear Information System (INIS)

    Zaleska, M.M.; Erecinska, M.

    1987-01-01

    Active, high-affinity, sodium-dependent uptake of [ 14 C]-aminobutyric acid and of the acidic amino acid D-[ 3 H]-aspartate was inhibited by pretreatment of synaptosomes with neuraminidase from Vibrio cholerae. Inhibition was of a noncompetitive type and was related to the amount of sialic acid released. The maximum accumulation ratios of both amino acids (intracellular [amino acid]/extracellular [amino acid]) remained largely unaltered. Treatment with neuraminidase affected neither the synaptosomal energy levels nor the concentration of internal potassium. It is suggested that the γ-aminobutyric acid and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of the carrier proteins directly and not through modification of driving forces responsible for amino acid uptake

  12. Ascorbic acid transport and accumulation in human neutrophils

    International Nuclear Information System (INIS)

    Washko, P.; Rotrosen, D.; Levine, M.

    1989-01-01

    The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake

  13. Affinity resins as new tools for identifying target proteins of ascorbic acid.

    Science.gov (United States)

    Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

    2018-02-12

    l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

  14. Interference-Blind Microfluidic Sensor for Ascorbic Acid Determination by UV/vis Spectroscopy

    DEFF Research Database (Denmark)

    Bi, Hongyan; Oliveira Fernandes, Ana Carolina; Cardoso, Susana

    2016-01-01

    A microfluidic sensor is developed and targeted at specific ingredients determination in drug/food/beverage matrices. The surface of a serpentine polydimethylsiloxane (PDMS) microchannel is modified by enzyme via physisorption. When solutions containing target ingredients pass through...... the microfluidic channel, enzyme-catalyzed reaction occurs and only converts the target molecules to its products. The whole process is monitored by an end-channel UV/vis spectroscopic detection. Ascorbate oxidase and L-ascorbic acid (AA) are taken as enzyme-substrate model in this study to investigate......, specific, and accurate, and can be potentially used for fast quantification of ingredient in samples with complex matrix background. It is promising to be widely spread in food industry and quality control department...

  15. Anion sensing with a Lewis acidic BODIPY-antimony(v) derivative.

    Science.gov (United States)

    Christianson, Anna M; Gabbaï, François P

    2017-02-21

    We describe the synthesis of a BODIPY dye substituted with a Lewis acidic antimony(v) moiety. This compound, which has been fully characterized, shows a high affinity for small anions including fluoride and cyanide, the complexation of which elicits a fluorescence turn-on response.

  16. Novel 3-carboxy- and 3-phosphonopyrazoline amino acids as potent and selective NMDA receptor antagonists

    DEFF Research Database (Denmark)

    Conti, Paola; Pinto, Andrea; Tamborini, Lucia

    2010-01-01

    The design and synthesis of new N1-substituted 3-carboxy- and 3-phosphonopyrazoline and pyrazole amino acids that target the glutamate binding site of NMDA receptors are described. An analysis of the stereochemical requirements for high-affinity interaction with these receptors was performed. We...

  17. Aortic wall damage in mice unable to synthesize ascorbic acid.

    Science.gov (United States)

    Maeda, N; Hagihara, H; Nakata, Y; Hiller, S; Wilder, J; Reddick, R

    2000-01-18

    By inactivating the gene for L-gulono-gamma-lactone oxidase, a key enzyme in ascorbic acid synthesis, we have generated mice that, like humans, depend on dietary vitamin C. Regular chow, containing about 110 mg/kg of vitamin C, is unable to support the growth of the mutant mice, which require L-ascorbic acid supplemented in their drinking water (330 mg/liter). Upon withdrawal of supplementation, plasma and tissue ascorbic acid levels decreased to 10-15% of normal within 2 weeks, and after 5 weeks the mutants became anemic, began to lose weight, and die. Plasma total antioxidative capacities were approximately 37% normal in homozygotes after feeding the unsupplemented diet for 3-5 weeks. As plasma ascorbic acid decreased, small, but significant, increases in total cholesterol and decreases in high density lipoprotein cholesterol were observed. The most striking effects of the marginal dietary vitamin C were alterations in the wall of aorta, evidenced by the disruption of elastic laminae, smooth muscle cell proliferation, and focal endothelial desquamation of the luminal surface. Thus, marginal vitamin C deficiency affects the vascular integrity of mice unable to synthesize ascorbic acid, with potentially profound effects on the pathogenesis of vascular diseases. Breeding the vitamin C-dependent mice with mice carrying defined genetic mutations will provide numerous opportunities for systematic studies of the role of antioxidants in health and disease.

  18. Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors

    DEFF Research Database (Denmark)

    Damgaard, Maria; Al-Khawaja, Anas; Nittegaard-Nielsen, Mia

    2017-01-01

    -affinity binding and is furthermore reported as an allosteric modulator selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined......-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [(3)H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have...... δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a GHB high...

  19. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

    Science.gov (United States)

    Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

  20. Conversion of the low affinity ouabain-binding site of non-gastric H,K-ATPase into a high affinity binding site by substitution of only five amino acids.

    NARCIS (Netherlands)

    Qiu, L.Y.; Swarts, H.G.P.; Tonk, E.C.; Willems, P.H.G.M.; Koenderink, J.B.; Pont, J.J.H.H.M. de

    2006-01-01

    P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven

  1. Defining proximity relationships in the tertiary structure of the dopamine transporter. Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn(2+)-binding site

    DEFF Research Database (Denmark)

    Løland, Claus Juul; Norregaard, L; Gether, U

    1999-01-01

    , high affinity Zn(2+)-binding site. To achieve further insight into the tertiary organization of hDAT, we set out to identify additional residues involved in Zn(2+) binding and subsequently to engineer artificial Zn(2+)-binding sites. Ten aspartic acids and glutamic acids, predicted...

  2. High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6 pmol/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350 pmol/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

  3. Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

    NARCIS (Netherlands)

    Dalla, S.; Swarts, H.G.P.; Koenderink, J.B.; Dobler, S.

    2013-01-01

    Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of

  4. The labeling of unsaturated γ-hydroxybutyric acid by heavy isotopes of hydrogen: iridium complex-mediated H/D exchange by C─H bond activation vs reduction by boro-deuterides/tritides

    DEFF Research Database (Denmark)

    Marek, Ales; Pedersen, Martin Holst Friborg; Vogensen, Stine B.

    2016-01-01

    3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA ( 1 )) is a potent ligand for high-affinity γ-hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C...

  5. Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

    Science.gov (United States)

    Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

    2010-03-05

    Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP. (c) 2009 Elsevier Ltd. All rights reserved.

  6. Radiosynthesis and in vitro evaluation of 2-(N-alkyl-N-1'-11C-propyl)amino-5-hydroxytetralin analogs as high affinity agonists for dopamine D-2 receptors

    International Nuclear Information System (INIS)

    Shi Bingzhi; Narayanan, Tanjore K.; Yang, Z.-Y.; Christian, Bradley T.; Mukherjee, Jogeshwar

    1999-01-01

    We have developed radiotracers based on agonists that may potentially allow the in vivo assessment of the high affinity (HA) state of the dopamine D-2 receptors. The population of HA state, which is likely the functional state of the receptor, may be altered in certain diseases. We carried out radiosyntheses and evaluated the binding affinities, lipophilicity, and in vitro autoradiographic binding characteristics of three dopamine D-2 receptor agonists: (±)-2-(N,N-dipropyl)amino-5-hydroxytetralin (5-OH-DPAT), (±)-2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), and (±)-2-(N-cyclohexylethyl-N-propyl)amino-5-hydroxytetralin (ZYY-339). In 3 H-spiperone assays using rat striata, ZYY-339 exhibited subnanomolar affinity for D-2 receptor sites ( IC 50 =0.010 nM), PPHT was somewhat weaker ( IC 50 =0.65 nM), and 5-OH-DPAT exhibited the weakest affinity ( IC 50 =2.5 nM) of the three compounds. Radiosynthesis of these derivatives, 2-(N-propyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-5-OH-DPAT), 2-(N-phenethyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-PPHT), and 2-(N-cyclohexylethyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-ZYY-339) was achieved by first synthesizing 11 C-1-propionyl chloride and subsequent coupling with the appropriate secondary amine precursor to form the respective amide, which was then reduced to provide the desired tertiary amine products. The final products were obtained by reverse-phase high performance liquid chromatography (HPLC) purification in radiochemical yields of 5-10% after 60-75 min from the end of 11 CO 2 trapping and with specific activities in the range of 250-1,000 Ci/mmol. In vitro autoradiographs in rat brain slices with 11 C-5-OH-DPAT, 11 C-PPHT, and 11 C-ZYY-339 revealed selective binding of the three radiotracers to the dopamine D-2 receptors in the striata

  7. (D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. (Department of Pharmacology, University of Arizona, College of Medicine, Tucson (United States))

    1991-09-01

    (D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

  8. Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain*

    Science.gov (United States)

    Bhattacharya, Sarbani; Liang, Zhong; Quek, Adam J.; Ploplis, Victoria A.; Law, Ruby; Castellino, Francis J.

    2014-01-01

    A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1–2 nm). However, addition of two PAM residues (Arg126-His127) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg113, His114, Glu116, Arg126, His127), mutation of which reduced PAM binding affinity for K2hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence. PMID:24962580

  9. Dimerization is not a determining factor for functional high affinity human plasminogen binding by the group A streptococcal virulence factor PAM and is mediated by specific residues within the PAM a1a2 domain.

    Science.gov (United States)

    Bhattacharya, Sarbani; Liang, Zhong; Quek, Adam J; Ploplis, Victoria A; Law, Ruby; Castellino, Francis J

    2014-08-01

    A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97-125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83-145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1-2 nm). However, addition of two PAM residues (Arg(126)-His(127)) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg(113), His(114), Glu(116), Arg(126), His(127)), mutation of which reduced PAM binding affinity for K2hPg by ∼ 1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3 H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 μg AAF/106 cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 × 10-7  M and VMAX[APPARENT] = 0.1 nmol/106 cells/day and System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 × 10-5  M and VMAX[APPARENT] = 1000 nmol/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 × 10-5  M and 4.7 nmol/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

  11. High- and low-affinity binding of S-citalopram to the human serotonin transporter mutated at 20 putatively important amino acid positions

    DEFF Research Database (Denmark)

    Plenge, Per; Wiborg, Ove

    2005-01-01

    of presumed importance. Binding of S-citalopram, both to the high-affinity-binding site and to the allosteric binding site, was measured in these mutants with the purpose of investigating the connection between the two binding sites. The amino acid substitutions did not introduce large changes in the two...

  12. Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces

    NARCIS (Netherlands)

    Sankaran, Shrikrishnan; Cavatorta, Emanuela; Huskens, Jurriaan; Jonkheijm, Pascal

    2017-01-01

    Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form

  13. 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

  14. Synthesis and pre-clinical evaluation of a new class of high-affinity "1"8F-labeled PSMA ligands for detection of prostate cancer by PET imaging

    International Nuclear Information System (INIS)

    Kelly, James; Amor-Coarasa, Alejandro; Williams, Clarence; Ponnala, Shashikanth; Nikolopoulou, Anastasia; Kim, Dohyun; Babich, John W.

    2017-01-01

    Current clinical imaging of PSMA-positive prostate cancer by positron emission tomography (PET) mainly features "6"8Ga-labeled tracers, notably ["6"8Ga]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 ["1"8F]triazoles were isolated following a Cu(I)-catalyzed click reaction between the alkynes and ["1"8F]fluoroethylazide. The "1"8F-labeled compounds were evaluated in nude mice bearing LNCaP tumors and compared to ["6"8Ga]Ga-PSMA-HBED-CC and ["1"8F]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_5_0) 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 ["6"8Ga]Ga-PSMA-HBED-CC and ["1"8F]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 ["6"8Ga]Ga-PSMA-HBED-CC. Six ["1"8F]triazolylphenyl ureas were prepared in good radiochemical yield

  15. Effect of intrahippocampal kainic acid injections and surgical lesions on neurotransmitters in hippocampus and septum

    Energy Technology Data Exchange (ETDEWEB)

    Fonnum, F; Walaas, I

    1978-01-01

    Local injection of kainic acid (2 ..mu..g) was accompanied by destruction of intrinsic neurons in the dorsal part of hippocampus. The lesion was accompanied by a 75% reduction in glutamate decarboxylase activity, a 60% reduction in the high affinity uptake of L-glutamate, a 40 to 60% reduction in the endogeneous levels of aspartate, glutamate and GABA and no changes in the activities of choline acetyltransferase or aromatic amino acid decarboxylase in the dorsal hippocampus. Unilateral destruction of neurons in the dorsal hippocampus was followed by a 20 to 40% reduction in the high affinity uptake of glutamate in lateral, but not in medial septum, on both sides. There was no reduction in choline acetyltransferase, glutamate decarboxylase or aromatic amino acid decarboxylase activities in the lateral or medial part of the septum. Transection of fimbria and superior fornix was accompanied by a severe reduction in choline acetyltransferase and aromatic amino acid decarboxylase activity in hippocampus, in the high affinity uptake of glutamate and in the endogenous level of glutamate in the lateral septum. The results are consistent with the concept that in the hippocampus kainic acid destroys intrinsic neurons and not afferent fibres. It seems therefore that all GABAergic fibres in the hippocampus belong to intrinsic neurons whereas glutamergic and aspartergic neurons belong partly to local neurons. The connection from the hippocampus to the lateral septum probably uses glutamate as a transmitter.

  16. Scaffolding along Nucleic Acid Duplexes Using 2'-Amino-Locked Nucleic Acids

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2014-01-01

    -LNA nucleotides. By application of different chemical reactions, modification of 2'-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2'-amino-LNA mainly....../DNA probes bind nucleic acid targets with advantages of high affinity and specificity. Thus, molecular motion of nanodevices and programmable self-assembly of chemically modified LNA/DNA nanomaterials can be followed by bright fluorescence signaling from the functionalized LNA units. Another appealing aspect...

  17. Nucleic Acid Aptamers Against Biotoxins: A New Paradigm Toward the Treatment and Diagnostic Approach

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Veedu, Rakesh N.

    2012-01-01

    Nucleic acid aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with very high affinity and specificity, and are generally selected by a process referred to as systematic evolution of ligands by exponential enrichment. Conventional antibody-based therape......Nucleic acid aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with very high affinity and specificity, and are generally selected by a process referred to as systematic evolution of ligands by exponential enrichment. Conventional antibody......-based therapeutic and diagnostic approach currently employed against biotoxins pose major limitations such as the requirement of a live animal for the in vivo enrichment of the antibody species, decreased stability, high production cost, and side effects. Aptamer technology is a viable alternative that can be used...

  18. Rational Design, Synthesis and Pharmacological Evaluation of the (2R)- and (2S)-Stereoisomers of 3-(2-Carboxypyrrolidinyl)-2-methyl Acetic Acid as Ligands for the Ionotropic Glutamate Receptors

    DEFF Research Database (Denmark)

    Rasmussen, Julie; Storgaard, Morten; Pickering, Darryl S

    2011-01-01

    In this paper we describe the rational design, synthesis and pharmacological evaluation of two new stereoisomeric (S)-glutamate (Glu) analogues. The rational design was based on hybrid structures of the natural product kainic acid, a synthetic analogue CPAA and the high-affinity Glu analogue SYM...

  19. Subtype selective kainic acid receptor agonists

    DEFF Research Database (Denmark)

    Bunch, Lennart; Krogsgaard-Larsen, Povl

    2009-01-01

    (S)-Glutamic acid (Glu) is the major excitatory neurotransmitter in the mammalian central nervous system, activating the plethora of glutamate receptors (GluRs). In broad lines, the GluRs are divided into two major classes: the ionotropic Glu receptors (iGluRs) and the metabotropic Glu receptors (m......GluRs). Within the iGluRs, five subtypes (KA1, KA2, iGluR5-7) show high affinity and express full agonist activity upon binding of the naturally occurring amino acid kainic acid (KA). Thus these receptors have been named the KA receptors. This review describes all-to our knowledge-published KA receptor agonists...

  20. Extraction of domoic acid from seawater and urine using a resin based on 2-(trifluoromethyl)acrylic acid.

    Science.gov (United States)

    Piletska, Elena V; Villoslada, Fernando Navarro; Chianella, Iva; Bossi, Alessandra; Karim, Kal; Whitcombe, Michael J; Piletsky, Sergey A; Doucette, Gregory J; Ramsdell, John S

    2008-03-03

    A new solid-phase extraction (SPE) matrix with high affinity for the neurotoxin domoic acid (DA) was designed and tested. A computational modelling study led to the selection of 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer capable of imparting affinity towards domoic acid. Polymeric adsorbents containing TFMAA were synthesised and tested in high ionic strength solutions such as urine and seawater. The TFMAA-based polymers demonstrated excellent performance in solid-phase extraction of domoic acid, retaining the toxin while salts and other interfering compounds such as aspartic and glutamic acids were removed by washing and selective elution. It was shown that the TFMAA-based polymer provided the level of purification of domoic acid from urine and seawater acceptable for its quantification by high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) without any additional pre-concentration and purification steps.

  1. Structural analysis and binding domain of albumin complexes with natural dietary supplement humic acid

    International Nuclear Information System (INIS)

    Ding Fei; Diao Jianxiong; Yang Xinling; Sun Ying

    2011-01-01

    Humic acid, a natural ionic molecule, is rapidly being recognized as one of the crucial elements in our modern diets of the new century. A biophysical protocol utilizing circular dichroism (CD), steady state and time-resolved fluorescence for the investigation of the complexation of the humic acid to the staple in vivo transporter, human serum albumin (HSA), as a model for protein-humic substances, is proclaimed. The alterations of CD and three-dimensional fluorescence suggest that the polypeptide chain of HSA partially folded after complexation with humic acid. The data of fluorescence emission displayed that the binding of humic acid to HSA is the formation of HSA-humic acid complex with an association constant of 10 4 M -1 ; this corroborates the fluorescence lifetime measurements that the static mechanism was operated. The precise binding domain of humic acid in HSA has been verified from the denaturation of albumin, hydrophobic ANS displacement, and site-specific ligands; subdomain IIA (Sudlow's site I) was earmarked to possess high-affinity for humic acid. The observations are relevant for other albumin-humic substance systems when the ligands have analogous configuration with humic acid. - Highlights: → Albumin structure partially folds upon humic acid complexation. → Static type is dominance for the diminution in the Trp-214 fluorescence.→ Subdomain IIA is designate to possess high-affinity site for humic acid.

  2. Bienzymatic Acetylcholinesterase and Choline Oxidase Immobilized Biosensor Based on a Phenyl Carboxylic Acid-Grafted Multiwalled Carbon Nanotube

    Directory of Open Access Journals (Sweden)

    So-Ra Lee

    2014-01-01

    Full Text Available Bienzymatic acetylcholinesterase (AChE and choline oxidase (ChOx immobilized biosensor based on a phenyl carboxylic acid-grafted multiwalled carbon nanotube (MWNT modified glass carbon electrode (GCE and carbon-screen printed electrode (SPE was fabricated for acetylcholine detection in human blood samples. Phenyl carboxylic acid-modified MWNT supports were prepared by electrochemical polymerization of 4-carboxyphenyl diazonium salts, which were synthesized by an amine group and sodium nitrite, on the surface of the MWNT-modified GCE and SPE in 0.1 M PBS. The successful fabrication of the AChE-ChOx-immobilized biosensor was confirmed via scanning electron microscopy (SEM, X-ray photoelectron spectroscopy (XPS, electrochemical impedance spectroscopy (EIS, and cyclic voltammetry (CV. The sensing range of the biosensor based on a GCE and SPE was 1.0~10 μM and 10~100 μM, respectively. The interfering effect of 0.1 M L-ascorbic acid, 0.1 M L-cysteine, and 0.1 M uric acid to 0.1 M acetylcholine was 3.00%, 9.00%, and 3.00%, respectively. Acetylcholine in a human blood sample was detected by the AChE-ChOx-immobilized biosensor.

  3. Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system.

    Science.gov (United States)

    Inoue, H; Hirobe, M

    1987-05-29

    The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.

  4. UlaR activates expression of the ula operon in Streptococcus pneumoniae in the presence of ascorbic acid.

    Science.gov (United States)

    Afzal, Muhammad; Shafeeq, Sulman; Henriques-Normark, Birgitta; Kuipers, Oscar P

    2015-01-01

    In this study, the regulatory mechanism of the ula (utilization of l-ascorbic acid) operon, putatively responsible for transport and utilization of ascorbic acid in Streptococcus pneumoniae strain D39, is studied. β-Galactosidase assay data demonstrate that expression of the ula operon is increased in the presence of ascorbic acid as compared with the effects of other sugar sources including glucose. The ula operon consists of nine genes, including a transcriptional regulator UlaR, and is transcribed as a single transcriptional unit. We demonstrate the role of the transcriptional regulator UlaR as a transcriptional activator of the ula operon in the presence of ascorbic acid and show that activation of the ula operon genes by UlaR is CcpA-independent. Furthermore, we predict a 16 bp regulatory site (5'-AACAGTCCGCTGTGTA-3') for UlaR in the promoter region of ulaA. Deletion of the half or full UlaR regulatory site in PulaA confirmed that the UlaR regulatory site present in PulaA is functional. © 2015 The Authors.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-15

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  7. Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

    DEFF Research Database (Denmark)

    Vorum, H; Pedersen, A O; Honoré, B

    1992-01-01

    Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture...... of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...... and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture...

  8. Highly improved synthesis of gold nanobipyramids by tuning the concentration of hydrochloric acid

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Ying; Zhu, Jian; Li, Jianjun; Zhao, Junwu, E-mail: nanoptzhao@163.com [Xi’an Jiaotong University, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology (China)

    2016-07-15

    Fabrication of gold nanobipyramids (Au BPs) has attracted great attention because they exhibit more advantageous plasmonic properties. In this study, Au BPs were synthesized by the well-known seeded growth in the presence of hydrochloric acid (HCl). The effects of the ingredients, including HCl, silver nitrate (AgNO{sub 3}), l-Ascorbic acid (AA), and seeds on the structure and yield of the Au BPs were systemically investigated. The results showed that the abundant HCl could improve the yield of Au BPs and decrease the longitudinal surface plasmon resonance wavelength. Under the circumstance of higher concentration of AA and AgNO{sub 3}, more byproducts were resulted. In addition, the effect of HCl under different ratios of seed solution to AA has also been studied. The results showed that the yield was less sensitive to HCl when the amount of seed solution was small. If substantial AA was added to the system, then abundant HCl should be introduced correspondingly to improve the yield of Au BPs.

  9. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    Science.gov (United States)

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  10. A novel fluorescence biosensor for sensitivity detection of tyrosinase and acid phosphatase based on nitrogen-doped graphene quantum dots.

    Science.gov (United States)

    Qu, Zhengyi; Na, Weidan; Liu, Xiaotong; Liu, Hua; Su, Xingguang

    2018-01-02

    In this paper, we developed a sensitive fluorescence biosensor for tyrosinase (TYR) and acid phosphatase (ACP) activity detection based on nitrogen-doped graphene quantum dots (N-GQDs). Tyrosine could be catalyzed by TYR to generate dopaquinone, which could efficiently quench the fluorescence of N-GQDs, and the degree of fluorescence quenching of N-GQDs was proportional to the concentration of TYR. In the presence of ACP, l-Ascorbic acid-2-phosphate (AAP) was hydrolyzed to generate ascorbic acid (AA), and dopaquinone was reduced to l-dopa, resulting in the fluorescence recovery of the quenched fluorescence by dopaquinone. Thus, a novel fluorescence biosensor for the detection of TYR and ACP activity based on N-GQDs was constructed. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of TYR and ACP in the range of 0.43-3.85 U mL -1 and 0.04-0.7 mU mL -1 with a detection limit of 0.15 U mL -1 and 0.014 mU mL -1 , respectively. The feasibility of the proposed biosensor in real samples assay was also studied and satisfactory results were obtained. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg²⁺ binding to the MIDAS.

    Science.gov (United States)

    Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J; Filizola, Marta; Springer, Timothy A; Coller, Barry S

    2012-03-14

    An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.

  12. Tritium-labelled leukotriene B4 binding to the guinea-pig spleen membrane preparation: a rich tissue source for a high-affinity leukotriene B4 receptor site

    International Nuclear Information System (INIS)

    Cheng, J.B.; Cheng, E.I.; Kohi, F.; Townley, R.G.

    1986-01-01

    Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [ 3 H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compete with [ 3 H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [ 3 H]LTB4 binding, but NaCl and KCl decreased it. Spleen [ 3 H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25 0 C was 0.47 nM-1 min-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg of protein. Moreover, the LTB4/[ 3 H]LTB4 competition study performed at 4 or 25 0 C revealed the inhibitory constant (Ki) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [ 3 H]LTB4 binding was: LTB4 (Ki = 2.8 nM) greater than 20-hydroxy-LTB4 (23 nM) greater than LTA4 (48 nM) greater than LTA4 methyl ester (0.13 microM) greater than 20-carboxy-LTB4 (greater than 6.6 microM) greater than or equal to arachidonic acid (0.15mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihyropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [ 3 H]LTB4 binding

  13. [(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

    Science.gov (United States)

    Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

    2002-02-11

    This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

  14. Fatty acid uptake in normal human myocardium

    International Nuclear Information System (INIS)

    Vyska, K.; Meyer, W.; Stremmel, W.; Notohamiprodjo, G.; Minami, K.; Machulla, H.J.; Gleichmann, U.; Meyer, H.; Koerfer, R.

    1991-01-01

    Fatty acid binding protein has been found in rat aortic endothelial cell membrane. It has been identified to be a 40-kDa protein that corresponds to a 40-kDa fatty acid binding protein with high affinity for a variety of long chain fatty acids isolated from rat heart myocytes. It is proposed that this endothelial membrane fatty acid binding protein might mediate the myocardial uptake of fatty acids. For evaluation of this hypothesis in vivo, influx kinetics of tracer-labeled fatty acids was examined in 15 normal subjects by scintigraphic techniques. Variation of the plasma fatty acid concentration and plasma perfusion rate has been achieved by modulation of nutrition state and exercise conditions. The clinical results suggest that the myocardial fatty acid influx rate is saturable by increasing fatty acid plasma concentration as well as by increasing plasma flow. For analysis of these data, functional relations describing fatty acid transport from plasma into myocardial tissue in the presence and absence of an unstirred layer were developed. The fitting of these relations to experimental data indicate that the free fatty acid influx into myocardial tissue reveals the criteria of a reaction on a capillary surface in the vicinity of flowing plasma but not of a reaction in extravascular space or in an unstirred layer and that the fatty acid influx into normal myocardium is a saturable process that is characterized by the quantity corresponding to the Michaelis-Menten constant, Km, and the maximal velocity, Vmax, 0.24 ± 0.024 mumol/g and 0.37 ± 0.013 mumol/g(g.min), respectively. These data are compatible with a nondiffusional uptake process mediated by the initial interaction of fatty acids with the 40-kDa membrane fatty acid binding protein of cardiac endothelial cells

  15. Novel (Phenylethynyl)pyrene-LNA Constructs for Fluorescence SNP Sensing in Polymorphic Nucleic Acid Targets

    DEFF Research Database (Denmark)

    Astakhova, Irina Kira; Samokhina, Evgeniya; Babu, B Ravindra

    2012-01-01

    We describe fluorescent oligonucleotide probes labeled with novel (phenylethynyl)pyrene dyes attached to locked nucleic acids. Furthermore, we prove the utility of these probes for the effective detection of single-nucleotide polymorphisms in natural nucleic acids. High-affinity hybridization......DNA and RNA gene fragments. Target sequences were obtained by analysis of 200 clinical samples from patients currently receiving anti-HIV/AIDS combination therapy at the Russian Federal AIDS Center. Using these fluorescent oligonucleotides, we were able to detect the target mutation despite all the challenges...

  16. Sugar-Responsive Pseudopolyrotaxane Composed of Phenylboronic Acid-Modified Polyethylene Glycol and γ-Cyclodextrin

    Directory of Open Access Journals (Sweden)

    Tomohiro Seki

    2015-03-01

    Full Text Available We have designed a sugar-responsive pseudopolyrotaxane (PPRX by combining phenylboronic acid-modified polyethylene glycol (PBA–PEG and γ-cyclodextrin. Phenylboronic acid (PBA was used as a sugar-recognition motif in the PPRX because PBA reacts with a diol portion of the sugar molecule and forms a cyclic ester. When D-fructose or D-glucose was added to a suspension of PPRX, PPRX disintegrated, depending on the concentration of the sugars. Interestingly, catechol does not show a response although catechol has a high affinity for PBA. We analyzed the response mechanism of PPRX by considering equilibria.

  17. Simultaneous Chronoamperometric Sensing of Ascorbic Acid and Acetaminophen at a Boron-Doped Diamond Electrode

    Directory of Open Access Journals (Sweden)

    Ciprian Radovan

    2008-06-01

    Full Text Available Cyclic voltammetry (CV and chronoamperometry (CA have been used to sense and determine simultaneously L-ascorbic acid (AA and acetaminophen (AC at a boron-doped diamond electrode (BDDE in a Britton-Robinson buffer solution. The calibration plots of anodic current peak versus concentration obtained from CV and CA data for both investigated compounds in single and di-component solutions over the concentration range 0.01 mM – 0.1 mM proved to be linear, with very good correlation parameters. Sensitivity values and RSD of 2-3% were obtained for various situations, involving both individual and simultaneous presence of AA and AC. The chronoamperometric technique associated with standard addition in sequential one step and/or two successive and continuous chronoamperograms at two characteristic potential levels represented a feasible option for the simultaneous determination of AA and AC in real sample systems such as pharmaceutical formulations. The average values indicated by the supplier were confirmed to a very close approximation from chronoamperomgrams by using several additions with the application of suitable current correction factors.

  18. Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism.

    Science.gov (United States)

    Parsons, Harriet T; Yasmin, Tayyaba; Fry, Stephen C

    2011-12-15

    L-Ascorbate catabolism involves reversible oxidation to DHA (dehydroascorbic acid), then irreversible oxidation or hydrolysis. The precursor-product relationships and the identity of several major DHA breakdown products remained unclear. In the presence of added H2O2, DHA underwent little hydrolysis to DKG (2,3-dioxo-L-gulonate). Instead, it yielded OxT (oxalyl L-threonate), cOxT (cyclic oxalyl L-threonate) and free oxalate (~6:1:1), essentially simultaneously, suggesting that all three product classes independently arose from one reactive intermediate, proposed to be cyclic-2,3-O-oxalyl-L-threonolactone. Only with plant apoplastic esterases present were the esters significant precursors of free oxalate. Without added H2O2, DHA was slowly hydrolysed to DKG. Downstream of DKG was a singly ionized dicarboxy compound (suggested to be 2-carboxy-L-xylonolactone plus 2-carboxy-L-lyxonolactone), which reversibly de-lactonized to a dianionic carboxypentonate. Formation of these lactones and acid was minimized by the presence of residual unreacted ascorbate. In vivo, the putative 2-carboxy-L-pentonolactones were relatively stable. We propose that DHA is a branch-point in ascorbate catabolism, being either oxidized to oxalate and its esters or hydrolysed to DKG and downstream carboxypentonates. The oxidation/hydrolysis ratio is governed by reactive oxygen species status. In vivo, oxalyl esters are enzymatically hydrolysed, but the carboxypentonates are stable. The biological roles of these ascorbate metabolites invite future exploration.

  19. Radioimmunoassay for the determination of free and conjugated abscisic acid

    International Nuclear Information System (INIS)

    Weiler, E.W.

    1979-01-01

    The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported, The antibodies produced against a bovine serum albumin-(+-)-ABA conjugate have a high affinity for ABA (Ka= 1.3 x 10 9 l mol -1 ). Trans, trans-ABA and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25 x 10 -12 mol ABA, the measuring range extends to 20 x 10 -12 mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi. (orig.) [de

  20. A High Affinity Adenosine Kinase from Anopheles gambiae

    Science.gov (United States)

    Cassera, María B.; Ho, Meng-Chiao; Merino, Emilio F.; Burgos, Emmanuel S.; Rinaldo-Matthis, Agnes; Almo, Steven C.; Schramm, Vern L.

    2011-01-01

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (Km 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap4A (2.0 Å resolution) reveals interactions for adenosine, ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg2+ ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layered α/β/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight-binding for adenosine arises from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168 and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64 and Asn68 and the ribosyl 2′- and 3′-hydroxyl groups. The structure is more similar to human adenosine kinase (48% identity) than to AK from Toxoplasma gondii (31% identity). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role of this enzyme to maintain the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects. PMID:21247194

  1. Photoaffinity labelling of high affinity dopamine binding proteins

    International Nuclear Information System (INIS)

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-01-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-]N'-4-azidobenzamidol]-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using ( 3 H)-SCH 23390 (a D 1 specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked ( 3 H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of ( 3 H)-SCH 23390 binding. Compounds which compete for D 1 receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D 1 (adenylate cyclase linked) dopamine receptor

  2. Antioxidant mechanism of milk mineral-high-affinity iron binding.

    Science.gov (United States)

    Allen, K; Cornforth, D

    2007-01-01

    Milk mineral (MM), a by-product of whey processing, is an effective antioxidant in meat systems, but the antioxidant mechanism has not been established. MM has been postulated to chelate iron and prevent iron-catalysis of lipid oxidation. The objective of this research was to examine this putative mechanism. MM was compared to sodium tripolyphosphate (STPP), calcium phosphate monobasic (CPM), and calcium pyrophosphate (CPP) to determine iron-binding capacity, sample solubility, and eluate soluble phosphorus after treating samples with a ferrous chloride standard. Scanning electron microscopy with energy-dispersive X-ray analysis was used to localize minerals on iron-treated MM particle surfaces. Histochemical staining for calcium was performed on raw and cooked ground beef samples with added MM. MM bound more iron per gram (P compounds, and was much less soluble (P iron across the MM particle surface, directly demonstrating iron binding to MM particles. Unlike other common chelating agents, such as STPP and citrate, histochemical staining demonstrated that MM remained insoluble in ground beef, even after cooking. The ability of MM to bind iron and remain insoluble may enhance its antioxidant effect by removing iron ions from solution. However, MM particles must be small and well distributed in order to adequately bind iron throughout the food system.

  3. Carrier-mediated γ-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

    2012-06-01

    The aim of the present study was to investigate the transport of γ-aminobutyric acid (GABA) across the basolateral membrane of intestinal cells. The proton-coupled amino acid transporter, hPAT1, mediates the influx of GABA and GABA mimetic drug substances such as vigabatrin and gaboxadol and the anticancer prodrug δ-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least two transporters seem to be involved in the basolateral transport of GABA. The basolateral uptake consisted of a high-affinity system with a K(m) of 290 μM and V(max) of 75 pmol cm(-2) min(-1) and a low affinity system with a K(m) of approximately 64 mM and V(max) of 1.6 nmol cm(-2) min(-1). The high-affinity transporter is Na(+) and Cl(-) dependent. The substrate specificity of the high-affinity transporter was further studied and Gly-Sar, Leucine, gaboxadol, sarcosine, lysine, betaine, 5-hydroxythryptophan, proline and glycine reduced the GABA uptake to approximately 44-70% of the GABA uptake in the absence of inhibitor. Other substances such as β-alanine, GABA, 5-aminovaleric acid, taurine and δ-aminolevulinic acid reduced the basolateral GABA uptake to 6-25% of the uptake in the absence of inhibitor. Our results indicate that the distance between the charged amino- and acid-groups is particular important for inhibition of basolateral GABA uptake. Thus, there seems to be a partial substrate overlap between the basolateral GABA transporter and hPAT1, which may prove important for understanding drug interactions at the level of intestinal transport. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Exploration of the dopamine transporter: in vitro and in vivo characterization of a high-affinity and high-specificity iodinated tropane derivative (E)-N-(3-iodoprop-2-enyl)-2β-carbomethoxy- 3β-(4'-methylphenyl)nortropane (PE2I)

    International Nuclear Information System (INIS)

    Guilloteau, Denis; Emond, Patrick; Baulieu, Jean-Louis; Garreau, Lucette; Frangin, Yves; Pourcelot, Leandre; Mauclaire, Laurent; Besnard, Jean-Claude; Chalon, Sylvie

    1998-01-01

    For the diagnosis and follow-up of neurodegenerative diseases, many cocaine derivatives have been proposed as radioligands to explore the dopamine transporter. As none of them have all the criteria of specificity and kinetics for human use, we have developed a new derivative, (E)-N-(3-iodoprop-2-enyl)-2β-carbomethoxy-3β-(4'-methylphenyl)nortropane (PE2I), which displays promising properties. We report the characterization of PE2I in vitro on rat striatal membranes and in vivo in rats and in monkeys. PE2I had a high affinity (Kd=0.09±0.01 nM) and high specificity for the dopamine transporter. In rats we observed a high accumulation in the striatum; by contrast, a very low fixation was measured in the cortex. Moreover, a preinjection of a saturating dose of GBR 12909 prevented the striatal accumulation of PE2I by 74%. These results confirmed the specificity of PE2I for the dopamine transporter. In vivo in monkeys, SPECT studies showed a high accumulation in striatum. Moreover, an equilibrium state was obtained 1 h after injection. PE2I seemed to be the most promising ligand for the dopamine transporter exploration by SPECT using a single-day protocol.

  5. Production of ascorbic acid releasing biomaterials for pelvic floor repair.

    Science.gov (United States)

    Mangır, Naşide; Bullock, Anthony J; Roman, Sabiniano; Osman, Nadir; Chapple, Christopher; MacNeil, Sheila

    2016-01-01

    An underlying abnormality in collagen turnover is implied in the occurrence of complications and recurrences after mesh augmented pelvic floor repair surgeries. Ascorbic acid is a potent stimulant of collagen synthesis. The aim of this study is to produce ascorbic acid releasing poly-lactic acid (PLA) scaffolds and evaluate them for their effects on extracellular matrix production and the strength of the materials. Scaffolds which contained either l-ascorbic acid (AA) and Ascorbate-2-Phosphate (A2P) were produced with emulsion electrospinning. The release of both drugs was measured by UV spectrophotometry. Human dermal fibroblasts were seeded on scaffolds and cultured for 2weeks. Cell attachment, viability and total collagen production were evaluated as well as mechanical properties. No significant differences were observed between AA, A2P, Vehicle and PLA scaffolds in terms of fibre diameter and pore size. The encapsulation efficiency and successful release of both AA and A2P were demonstrated. Both AA and A2P containing scaffolds were significantly more hydrophilic and stronger in both dry and wet states compared to PLA scaffolds. Fibroblasts produced more collagen on scaffolds containing either AA or A2P compared to cells grown on control scaffolds. This study is the first to directly compare the two ascorbic acid derivatives in a tissue engineered scaffold and shows that both AA and A2P releasing electrospun PLA scaffolds increased collagen production of fibroblasts to similar extents but AA scaffolds seemed to be more hydrophilic and stronger compared to A2P scaffolds. Mesh augmented surgical repair of the pelvic floor currently relies on non-degradable materials which results in severe complications in some patients. There is an unmet and urgent need for better pelvic floor repair materials. Our current understanding suggests that the ideal material should be able to better integrate into sites of implantation both biologically and mechanically. The impact of

  6. Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4

    DEFF Research Database (Denmark)

    Watterson, Kenneth R; Hansen, Steffen V F; Hudson, Brian D

    2017-01-01

    High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential...... of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many...

  7. Nucleic Acid Aptamers as Novel Class of Therapeutics to Mitigate Alzheimer's Disease Pathology

    DEFF Research Database (Denmark)

    K. Tannenberg, Rudi; Al. Shamaileh, Hadi; Lauridsen, Lasse Holm

    2013-01-01

    Deposition of amyloid-beta (A beta) peptides in the brain is a central event in the pathogenesis of Alzheimer's disease (AD), which makes A beta peptides a crucial target for therapeutic intervention. Significant efforts have been made towards the development of ligands that bind to A beta peptides...... with a goal of early detection of amyloid aggregation and the neutralization of A toxicity. Short single-stranded oligonucleotide aptamers bind with high affinity and specificity to their targets. Aptamers that specifically bind to A beta monomers, specifically the 40 and 42 amino acid species (A beta(1...

  8. All-solid-state potentiometric sensors for ascorbic acid by using a screen-printed compatible solid contact

    International Nuclear Information System (INIS)

    Veltsistas, Panayotis G.; Prodromidis, Mamas I.; Efstathiou, Constantinos

    2004-01-01

    The development of all-solid-state potentiometric ion selective electrodes for monitoring of ascorbic acid, by using a screen-printed compatible solid contact is described. The applied methodology is based on the use of PVC membrane modified with some firstly-tested ionophores (triphenyltin(IV)chloride, triphenyltin(IV)hydroxide and palmitoyl-L-ascorbic acid) and a novel one synthesized in our laboratory (dibutyltin(IV) diascorbate). Synthesis protocol and some preliminary identification studies are given. A conductive graphite-based polymer thick film ink was used as an internal solid contact between the graphite electrode and the PVC membrane. The presence and the nature of the solid contact (plain or doped with lanthanum 2,6-dichlorophenolindophenol (DCPI)) seem to enhance the analytical performance of the electrodes in terms of sensitivity, dynamic range, and response time. The analytical performance of the constructed electrodes was evaluated with potentiometry, constant-current chronopotentiometry and electrochemical impedance spectroscopy (EIS). The interference effect of various compounds was also tested. The potential response of the optimized Ph 3 SnCl-based electrode was linear against ascorbic acid concentration range 0.005-5.0 mM. The applicability of the proposed sensors in real samples was also tested. The detection limit was 0.002 mM ascorbic acid (50 mM phosphate, pH 5 in 50 mM KCl). The slope of the electrodes was super-Nernstian and pH dependent, indicating a mechanism involving a combination of charge transfer and ion exchange processes. Fabrication of screen-printed ascorbate ISEs has also been demonstrated

  9. Immobilization of uricase on ZnO nanorods for a reagentless uric acid biosensor

    International Nuclear Information System (INIS)

    Zhang Fenfen; Wang Xiaoli; Ai Shiyun; Sun Zhengdong; Wan Qiao; Zhu Ziqiang; Xian Yuezhong; Jin Litong; Yamamoto, Katsunobu

    2004-01-01

    A reagentless uric acid (UA) biosensor based on uricase immobilized on ZnO nanorods was developed. Direct electrochemistry and thermal stability of immobilized uricase were studied. The ZnO nanorods derived electrode retained the enzyme bioactivity and could enhance the electron transfer between the enzyme and the electrode. This sensor showed a high thermal stability up to 85 deg. C and an electrocatalytic activity to the oxidation of uric acid without the presence of an electron mediator. The electrocatalytic response showed a linear dependence on the uric acid concentration ranging from 5.0 x 10 -6 to 1.0 x 10 -3 mol L -1 with a detection limit of 2.0 x 10 -6 mol L -1 at 3σ. The apparent K M app value for the uric acid sensor was estimated to be 0.238 mM, showing a high affinity

  10. Bacterial fatty acid metabolism in modern antibiotic discovery.

    Science.gov (United States)

    Yao, Jiangwei; Rock, Charles O

    2017-11-01

    Bacterial fatty acid synthesis is essential for many pathogens and different from the mammalian counterpart. These features make bacterial fatty acid synthesis a desirable target for antibiotic discovery. The structural divergence of the conserved enzymes and the presence of different isozymes catalyzing the same reactions in the pathway make bacterial fatty acid synthesis a narrow spectrum target rather than the traditional broad spectrum target. Furthermore, bacterial fatty acid synthesis inhibitors are single-targeting, rather than multi-targeting like traditional monotherapeutic, broad-spectrum antibiotics. The single-targeting nature of bacterial fatty acid synthesis inhibitors makes overcoming fast-developing, target-based resistance a necessary consideration for antibiotic development. Target-based resistance can be overcome through multi-targeting inhibitors, a cocktail of single-targeting inhibitors, or by making the single targeting inhibitor sufficiently high affinity through a pathogen selective approach such that target-based mutants are still susceptible to therapeutic concentrations of drug. Many of the pathogens requiring new antibiotic treatment options encode for essential bacterial fatty acid synthesis enzymes. This review will evaluate the most promising targets in bacterial fatty acid metabolism for antibiotic therapeutics development and review the potential and challenges in advancing each of these targets to the clinic and circumventing target-based resistance. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Aspartic acid

    Science.gov (United States)

    ... we eat. Aspartic acid is also called asparaginic acid. Aspartic acid helps every cell in the body work. It ... release Normal nervous system function Plant sources of aspartic acid include: avocado, asparagus, and molasses. Animal sources of ...

  12. 2-Amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid

    DEFF Research Database (Denmark)

    Johansen, Tommy N; Janin, Yves L; Nielsen, Birgitte

    2002-01-01

    In order to identify new subtype-selective (S)-glutamate (Glu) receptor ligands we have synthesized (RS)-2-amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid [(RS)-TDPA]. Resolution of (RS)-TDPA by chiral chromatography was performed using a Crownpac CR(+) column affording (R)- and (S......)-TDPA of high enantiomeric purity (enantiomeric excess=99.9%). An X-ray crystallographic analysis revealed that the early eluting enantiomer has R-configuration. Both enantiomers showed high affinity as well as high agonist activity at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA...... a remarkably low AMPA receptor stereoselectivity, (S)-TDPA showing the highest affinity and (R)-TDPA the most potent agonist activity. In addition, (S)-TDPA was shown to interact with synaptosomal Glu uptake sites displacing [(3)H](R)-aspartic acid (IC(50 ) approximately 390 microM). An enantiospecific...

  13. Combining reduced glutathione and ascorbic acid has supplementary beneficial effects on boar sperm cryotolerance.

    Science.gov (United States)

    Giaretta, Elisa; Estrada, Efrén; Bucci, Diego; Spinaci, Marcella; Rodríguez-Gil, Joan E; Yeste, Marc

    2015-02-01

    The main aim of this work was to evaluate how supplementing freezing and thawing media with reduced glutathione (GSH) and l-ascorbic acid (AA) affected the quality parameters of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12 ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to which 5 mM of GSH and 100 μM of AA were added separately or together at two different steps of freeze-thawing. Various sperm parameters (levels of free cysteine residues in sperm nucleoproteins, sperm viability, acrosome membrane integrity, intracellular peroxide and superoxide levels [ROS], and total and progressive motility) were evaluated before freezing and at 30 and 240 minutes after thawing. Both GSH and AA significantly improved boar sperm cryotolerance when they were separately added to freezing and thawing media. However, the highest improvement was recorded when both freezing and thawing media were supplemented with 5 mM of GSH plus 100 μM of AA. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels and had no impact on superoxide levels. According to our results, we can conclude that supplementation of freezing and thawing media with both GSH and AA has a combined, beneficial effect on frozen-thawed boar sperm, which is greater than that obtained with the separate addition of either GSH or AA. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Effects of an ascorbic acid-derivative dentifrice in patients with gingivitis: a double-masked, randomized, controlled clinical trial.

    Science.gov (United States)

    Shimabukuro, Yoshio; Nakayama, Yohei; Ogata, Yorimasa; Tamazawa, Kaoru; Shimauchi, Hidetoshi; Nishida, Tetsuya; Ito, Koichi; Chikazawa, Takashi; Kataoka, Shinsuke; Murakami, Shinya

    2015-01-01

    Reactive oxygen species might be associated with the onset and progression of gingival inflammation. The aim of this study is to investigate the effect of a dentifrice containing L-ascorbic acid 2-phosphate magnesium salt (APM), a long-acting ascorbic acid derivative with antioxidant properties, on gingival inflammation. The clinical effects of APM were investigated in a multicenter, randomized, parallel-group, controlled clinical trial comprising 300 individuals with gingivitis. Half of the participants were given an APM-containing dentifrice and half were given a control dentifrice. The primary outcome was the gingival index (GI) at 3 months. Secondary outcomes included gingival redness as an indicator of the degree of local gingival inflammation, gingival bleeding as a measure of the gingivitis severity index, and total antioxidant activity of the saliva. Under the intent-to-treat analysis, GI did not significantly differ between the groups (P = 0.12). However, under the per-protocol analysis, GI was significantly lower in the APM group (P = 0.01) than in the control group. In the APM group, gingival redness was significantly lower, and the difference from the baseline gingivitis severity index was significantly greater (P = 0.04 and P = 0.02, respectively). The total antioxidant activity of the saliva was significantly higher in the APM group (P = 0.03). The incidence of adverse events did not significantly differ between the groups (P > 0.15). These findings indicate that the regular application of an APM-containing dentifrice could reduce gingival inflammation.

  15. Resolution, configurational assignment, and enantiopharmacology at glutamate receptors of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and demethyl-ACPA

    DEFF Research Database (Denmark)

    Johansen, T N; Stensbøl, T B; Nielsen, B

    2001-01-01

    We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution...... of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC...... columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC(50) = 0.025 microM), low affinity in kainic acid...

  16. Electrocatalytic activity of Cu2O nanocubes-based electrode for ...

    Indian Academy of Sciences (India)

    sensitive, selective, and fast amperometric sensing of glucose, which is promising for the future development of non-enzymatic ... sensors have attracted much attention because of their ... addition of 0.1 M L-ascorbic acid, the colour changed.

  17. Mineral, vitamin C and crude protein contents in kale (Brassica ...

    African Journals Online (AJOL)

    ajl yemi

    2011-10-27

    Oct 27, 2011 ... Key words: Kale (Brassica oleracea var. acephala), harvesting stage, vitamin C, crude protein, mineral content. .... L-ascorbic acid (or vitamin C) in plant tissues. .... Cooking methods of Brassica rapa affect the preservation of.

  18. Acid Rain

    Science.gov (United States)

    Bricker, Owen P.; Rice, Karen C.

    1995-01-01

    Although acid rain is fading as a political issue in the United States and funds for research in this area have largely disappeared, the acidity of rain in the Eastern United States has not changed significantly over the last decade, and it continues to be a serious environmental problem. Acid deposition (commonly called acid rain) is a term applied to all forms of atmospheric deposition of acidic substances - rain, snow, fog, acidic dry particulates, aerosols, and acid-forming gases. Water in the atmosphere reacts with certain atmospheric gases to become acidic. For example, water reacts with carbon dioxide in the atmosphere to produce a solution with a pH of about 5.6. Gases that produce acids in the presence of water in the atmosphere include carbon dioxide (which converts to carbonic acid), oxides of sulfur and nitrogen (which convert to sulfuric and nitric acids}, and hydrogen chloride (which converts to hydrochloric acid). These acid-producing gases are released to the atmosphere through natural processes, such as volcanic emissions, lightning, forest fires, and decay of organic matter. Accordingly, precipitation is slightly acidic, with a pH of 5.0 to 5.7 even in undeveloped areas. In industrialized areas, most of the acid-producing gases are released to the atmosphere from burning fossil fuels. Major emitters of acid-producing gases include power plants, industrial operations, and motor vehicles. Acid-producing gases can be transported through the atmosphere for hundreds of miles before being converted to acids and deposited as acid rain. Because acids tend to build up in the atmosphere between storms, the most acidic rain falls at the beginning of a storm, and as the rain continues, the acids "wash out" of the atmosphere.

  19. Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots.

    Science.gov (United States)

    Negrel, Jonathan; Javelle, Francine; Morandi, Dominique; Lucchi, Géraldine

    2016-12-01

    A Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (K m  = 2 μM) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid isomers), feruloyl esterases substrates (methyl caffeate and methyl ferulate), and even caffeoyl-CoA in vitro but all of them were less active than chlorogenic acid, demonstrating that the esterase is a genuine chlorogenic acid esterase. It was also induced when the bacterial strain was cultured in the presence of hydroxycinnamic acids (caffeic, p-coumaric or ferulic acid) as sole carbon source, but not in the presence of simple phenolics such as catechol or protocatechuic acid, nor in the presence of organic acids such as succinic or quinic acids. The purified esterase was remarkably stable in the presence of methanol, rapid formation of methyl caffeate occurring when its activity was measured in aqueous solutions containing 10-60% methanol. Our results therefore show that this bacterial chlorogenase can catalyse the transesterification reaction previously detected during the methanolic extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots. Data are presented suggesting that colonisation by Rhizophagus irregularis could increase chlorogenic acid exudation from tomato roots, especially in nutrient-deprived plants, and thus favour the growth of chlorogenate-metabolizing bacteria on the root surface or in the mycorhizosphere. Copyright © 2016 Elsevier Masson SAS. All rights

  20. Selective separation of indium by iminodiacetic acid chelating resin

    International Nuclear Information System (INIS)

    Fortes, M.C.B.; Benedetto, J.S.; Martins, A.H.

    2007-01-01

    - Indium can be recovered by treating residues, flue dusts, slags, and metallic intermediates in zinc smelting. This paper investigates the adsorption characteristics of indium and iron on an iminodiacetic acid chelating resin, Amberlite R IRC748 (Rohm and Haas Co.-USA). High concentrations of iron are always present in the aqueous feed solution of indium recovery. In addition, the chemical behaviour of iron in adsorptive systems is similar to that of indium. The metal concentrations in the aqueous solution were based on typical indium sulfate leach liquor obtained from zinc hydrometallurgical processing in a Brazilian plant. The ionic adsorption experiments were carried out by the continuous column method. Amberlite R IRC748 resin had a high affinity for indium under acidic conditions. Indium ions adsorbed onto the polymeric resin were eluted with a 0.5 mol/dm 3 sulphuric acid solution passed through the resin bed in the column. 99.5% pure indium sulfate aqueous solution was obtained using the iminodiacetic acid chelating resin Amberlite R IRC748. (author)

  1. Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid

    Energy Technology Data Exchange (ETDEWEB)

    Mertens, R.; Weiler, E.W. (Bochum Univ. (Germany, F.R.). Lehrstuhl fuer Pflanzenphysiologie); Deus-Neumann, B. (Muenchen Univ. (Germany, F.R.). Inst. fuer pharmazeutische Biologie)

    1983-08-22

    Monoclonal antibodies (mAB) have been produced which recognize the physiologically active 2-cis-(S)-form of the endogenous plant growth regulator, abscisic acid (ABA). Cross-reaction with the ABA-catabolites, phaseic and dihydrophaseic acid, is negligible, and (R)-ABA, 2-trans-ABA, the ABA-conjugate, ABA-..beta..-D-glucopyranosyl ester, as well as the putative ABA precursor, xanthoxin, are totally unreactive. In addition to being very specific, the mAB exhibit high affinities for 2-cis-(S)-ABA; the K values were 7.9 x 10/sup 9/ l/mol and 3.7 x 10/sup 9/ l/mol for antibodies from two different clones. By mAB-radioimmunoassay (RIA), 4 pg of 2-cis-(S)-ABA (99.5% confidence level) can be detected. mAB-RIA can be used to quantitate ABA directly in unprocessed plant extracts.

  2. Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid

    International Nuclear Information System (INIS)

    Mertens, R.; Weiler, E.W.; Deus-Neumann, B.

    1983-01-01

    Monoclonal antibodies (mAB) have been produced which recognize the physiologically active 2-cis-(S)-form of the endogenous plant growth regulator, abscisic acid (ABA). Cross-reaction with the ABA-catabolites, phaseic and dihydrophaseic acid, is negligible, and (R)-ABA, 2-trans-ABA, the ABA-conjugate, ABA-β-D-glucopyranosyl ester, as well as the putative ABA precursor, xanthoxin, are totally unreactive. In addition to being very specific, the mAB exhibit high affinities for 2-cis-(S)-ABA; the K values were 7.9 x 10 9 l/mol and 3.7 x 10 9 l/mol for antibodies from two different clones. By mAB-radioimmunoassay (RIA), 4 pg of 2-cis-(S)-ABA (99.5% confidence level) can be detected. mAB-RIA can be used to quantitate ABA directly in unprocessed plant extracts. (Auth.)

  3. Acidity, oxophilicity and hydrogen sticking probability of supported metal catalysts for hydrodeoxygenation process

    Science.gov (United States)

    Lup, A. Ng K.; Abnisa, F.; Daud, W. M. A. W.; Aroua, M. K.

    2018-03-01

    Hydrodeoxygenation is an oxygen removal process that occurs in the presence of hydrogen and catalysts. This study has shown the importance of acidity, oxophilicity and hydrogen sticking probability of supported metal catalysts in having high hydrodeoxygenation activity and selectivity. These properties are required to ensure the catalyst has high affinity for C-O or C=O bonds and the capability for the adsorption and activation of H2 and O-containing compounds. A theoretical framework of temperature programmed desorption technique was also discussed for the quantitative understanding of these properties. By using NH3-TPD, the nature and abundance of acid sites of catalyst can be determined. By using H2-TPD, the nature and abundance of metallic sites can also be determined. The desorption activation energy could also be determined based on the Redhead analysis of TPD spectra with different heating rates.

  4. Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose.

    Science.gov (United States)

    Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Petrescu, Anca D; Landrock, Kerstin K; Landrock, Danilo; Kier, Ann B; Schroeder, Friedhelm

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPAR α transcription of the fatty acid β -oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPAR α in the context of high glucose at levels similar to those in uncontrolled diabetes.

  5. The challenge of increasing vitamin C content in plant foods.

    Science.gov (United States)

    Cruz-Rus, Eduardo; Amaya, Iraida; Valpuesta, Victoriano

    2012-09-01

    The term "vitamin" is used to define a number of organic compounds that have to be obtained from different foods because the organism itself cannot synthesize them in the quantities needed to sustain life. Vitamin C is the common name for L-ascorbic acid. In humans, the principal role of this molecule is to scavenge reactive oxygen species, due to its antioxidant capacity, and to serve as cofactor for many enzymes. A deficiency of L-ascorbic acid is traditionally linked to human diseases such as scurvy. Plant foods are the principal source of L-ascorbic acid for humans. There is a high variability of L-ascorbic acid content in the various plant organs that are used for human consumption. This diversity is related to the specific functions played by L-ascorbic acid in the different plant tissues. The net content of L-ascorbic acid in plants is determined through a balance of the activities of different biosynthetic, recycling, and catabolic pathways. Here we review the importance of L-ascorbic acid for human health, the current knowledge on its metabolism and function in plants, and the efforts that have already been made by genetic modification to improve its content in plant organs used for human food. We provide a current and forward looking perspective of how plant science can contribute to improving the L-ascorbic acid content in crop species using gene transformation, quantitative trait loci and association mapping-based approaches. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Insulin binding and stimulation of hexose and amino acid transport by normal and receptor-defective human fibroblasts

    International Nuclear Information System (INIS)

    Longo, N.; Nagata, N.; Danner, D.; Priest, J.; Elsas, L.

    1986-01-01

    The authors analyzed insulin receptors in cells cultured from a sibship of related parents who had two offspring with severe insulin resistance (Leprechaunism). 124 I-Insulin (1 ng/ml) binding to skin fibroblasts from the proband, mother, and father was 9, 60 and 62% of control cells, respectively, at equilibrium, Non-linear regression analysis, utilizing a two receptors model, of curvilinear Scatchard plots indicated a reduced number of high-affinity binding sites in both parents. Influx of L-Proline (System A), L-Serine (ASC) and L-Leucine (L) was similar in control and mutant cells. Similarly, during the depletion of intracellular amino acid pools, there was a release from transinhibition for System A and a decrease of transstimulation of Systems ASC and L in both cell lines. Surprisingly, insulin augmented, normally, A system influx with an ED 50 = 70 ng/ml at 24 0 C and 7 ng/ml at 37 0 C. By contrast insulin failed to simulated 3-0-methyl-D-glucose influx into the proband's cells, while normal cells were stimulated 30% with an ED 50 of 6 ng/ml. These results indicate that defective high-affinity insulin binding is inherited as an autosomal recessive trait; that general membrane functions are intact; that insulin regulates A system amino acid and hexose transport by two different mechanisms; and, that the latter mechanism is impaired by this family's receptor mutation

  7. Thioesterase activity and acyl-CoA/fatty acid cross-talk of hepatocyte nuclear factor-4{alpha}.

    Science.gov (United States)

    Hertz, Rachel; Kalderon, Bella; Byk, Tamara; Berman, Ina; Za'tara, Ghadeer; Mayer, Raphael; Bar-Tana, Jacob

    2005-07-01

    Hepatocyte nuclear factor-4alpha (HNF-4alpha) activity is modulated by natural and xenobiotic fatty acid and fatty acyl-CoA ligands as a function of their chain length, unsaturation, and substitutions. The acyl-CoA site of HNF-4alpha is reported here to consist of the E-F domain, to bind long-chain acyl-CoAs but not the respective free acids, and to catalyze the hydrolysis of bound fatty acyl-CoAs. The free acid pocket, previously reported in the x-ray structure of HNF-4alpha E-domain, entraps fatty acids but excludes acyl-CoAs. The acyl-CoA and free acid sites are distinctive and noncongruent. Free fatty acid products of HNF-4alpha thioesterase may exchange with free acids entrapped in the fatty acid pocket of HNF-4alpha. Cross-talk between the acyl-CoA and free fatty acid binding sites is abrogated by high affinity, nonhydrolyzable acyl-CoA ligands of HNF-4alpha that inhibit its thioesterase activity. Hence, HNF-4alpha transcriptional activity is controlled by its two interrelated acyl ligands and two binding sites interphased in tandem by the thioesterase activity. The acyl-CoA/free-acid and receptor/enzyme duality of HNF-4alpha extends the paradigm of nuclear receptors.

  8. Study of the protective effect of ascorbic acid against the toxicity of stannous chloride on oxidative damage, antioxidant enzymes and biochemical parameters in rabbits.

    Science.gov (United States)

    Yousef, M I; Awad, T I; Elhag, F A; Khaled, F A

    2007-06-25

    Stannous chloride (SnCl2) is a reducing chemical agent used in several man-made products. SnCl2 can generate reactive oxygen species (ROS). Therefore, the present study has been carried out to investigate the antioxidant action of l-ascorbic acid (AA) in minimizing SnCl2 toxicity on lipid peroxidation, antioxidant enzyme, and biochemical parameters in male New Zealand white rabbits. Animals were assigned to one of four treatment groups: 0mg AA and 0mg SnCl2/kg BW (control); 40 mg AA/kg BW; 20mg SnCl2/kg BW; 20mg SnCl2 plus 40 mg AA/kg BW. Rabbits were orally administered the respective doses every other day for 12 weeks. Results obtained showed that SnCl2 significantly (Pacid-reactive substances (TBARS; the marker of lipid peroxidation) in plasma, while the activities of glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT), and the level of sulfhydryl groups (SH-group) were decreased (Pacid phosphatase (AcP) and lactate dehydrogenase (LDH) activities were decreased (Pcholesterol, triglyceride (TG), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL), glucose, urea and total bilirubin. On the other hand, the level of plasma high-density lipoprotein (HDL), total protein (TP), albumin (A) and globulin (G) were significantly (PAscorbic acid alone significantly decreased the levels of TBARS, lipids and urea, and increased the activities of GST, SOD and CAT, and the levels of SH-group and proteins. While the rest of the tested parameters were not affected. Also, the presence of AA with SnCl2 alleviated its harmful effects on most of the tested parameters. Therefore, the present results revealed that treatment with AA could minimize the toxic effects of stannous chloride.

  9. Amino acids

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/002222.htm Amino acids To use the sharing features on this page, please enable JavaScript. Amino acids are organic compounds that combine to form proteins . ...

  10. Valproic Acid

    Science.gov (United States)

    ... acid is in a class of medications called anticonvulsants. It works by increasing the amount of a ... older (about 1 in 500 people) who took anticonvulsants such as valproic acid to treat various conditions ...

  11. Ascorbic Acid

    Science.gov (United States)

    Ascorbic acid is used to prevent and treat scurvy, a disease caused by a lack of vitamin C in ... Ascorbic acid comes in extended-release (long-acting) capsules and tablets, lozenges, syrup, chewable tablets, and liquid drops to ...

  12. Aminocaproic Acid

    Science.gov (United States)

    Aminocaproic acid is used to control bleeding that occurs when blood clots are broken down too quickly. This type ... the baby is ready to be born). Aminocaproic acid is also used to control bleeding in the ...

  13. Ethacrynic Acid

    Science.gov (United States)

    Ethacrynic acid, a 'water pill,' is used to treat swelling and fluid retention caused by various medical problems. It ... Ethacrynic acid comes as a tablet to take by mouth. It is usually taken once or twice a day ...

  14. Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate its interaction with lipoprotein lipase.

    Science.gov (United States)

    Robal, Terje; Larsson, Mikael; Martin, Miina; Olivecrona, Gunilla; Lookene, Aivar

    2012-08-24

    Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.

  15. Isolation of amino acid activating subunit-pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis

    Science.gov (United States)

    Lee, Sung G.; Lipmann, Fritz

    1977-01-01

    Dissociation of the multienzymes of tyrocidine synthesis by prolonged incubation of crude extracts of Bacillus brevis (Dubos strain, ATCC 8185) has yielded, on Sephadex G-100 chromatography, two fractions of amino acid activating subunits, a larger one of 70,000 daltons and a smaller one of 90,000 daltons; the latter was a complex consisting of the 70,000 dalton subunit and the pantetheine-carrying protein of about 20,000 daltons. When it dissociated, the intermediate enzyme, which activates three amino acids, contained two-thirds of the subunits in the 70,000 dalton and one-third in the 90,000 dalton fraction; the heavy enzyme, which activates six amino acids, contained five-sixths of the subunits in the former fraction and one-sixth in the latter. Both fractions showed ATP-PPi exchange with all amino acids that are activated by the respective polyenzymes. With proline as an example, the 70,000 dalton subunit exhibited a single low-affinity binding site, which should correspond to the peripheral thiol acceptor site, whereas the 90,000 dalton subunit showed both a low-affinity binding site and an additional high-affinity site for proline; the high-affinity site is attributed to the pantetheine present on the pantetheine-carrying protein, and suggests that amino acids are translocated from the peripheral SH to the pantetheine-carrying moiety during chain elongation. This was confirmed by the observation that the 90,000 dalton complex, when incubated with the light enzyme in the presence of phenylalanine and proline, produced DPhe-Pro dipeptide that cyclized into DPhe-Pro diketopiperazine, but the 70,000 dalton activating subunit, when similarly incubated, did not. After subunit dissociation, however, no further elongation occurred after the transfer from phenylalanine to proline. Images PMID:196286

  16. Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

    Science.gov (United States)

    Fincham, D A; Ellory, J C; Young, J D

    1992-08-01

    In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target

  17. Analysis of long-chain fatty acid binding activity in vesicles of the outer membrane generated from Escherchia coli

    International Nuclear Information System (INIS)

    Black, P.N.

    1987-01-01

    Escherichia coli transports long-chain fatty acids across the dual membrane by a high affinity, saturable, energy-dependent process. The fadL gene codes for an outer membrane protein which appears to act specifically as a long-chain fatty acid binding protein when fatty acid utilization is blocked by mutation. In an effort to understand the function of the fadL gene product, FLP, membranes have been isolated from fadL + and fadL - strains following osmotic lysis. Following isolation, total membranes were separated into inner and outer membrane fractions and assayed for long-chain fatty acid binding activity. Outer membrane vesicles were incubated 2-5 min at 37 0 C with 3 H oleate (C/sub 18:1/), cooled to 0 0 C, and centrifuged through a Lipidex 100 column for 3 min to remove the unbound fatty acid. The level of fatty acid binding was quantitated by scintillation counting of the eluate. Outer membrane vesicles generated from a fadL + strain bind 325 pmol fatty acid/mg protein whereas vesicles generated for a mutant strain bind 175 pmol fatty acid/mg protein. These data suggest that FLP acts at least as a long-chain fatty acid binding protein on the surface of the cell

  18. Well acidizing

    Energy Technology Data Exchange (ETDEWEB)

    Street, E H

    1980-01-23

    The apparatus relates in particular to a well-treating process in which an aqueous acid solution having a pH of < 2 is injected into a subterranean reservoir in a manner such that materials that contain ferric ions are present in the acid and, as the acid reacts within the reservoir and attains a pH exceeding 3, tend to be precipitated as ferric ion-containing solid materials that may plug the pores of the reservoir. Such a precipitation is prevented by dissolving in the acid solution an amount of 5-sulfosalicylic acid which is at least sufficient to sequester significant proportions of ferric ions when the pH of the acid is from 0.5 to 3 but is less than enough to cause a significant salting-out of solid materials, and an amount of citric acid which is at least sufficient to sequester significant proportions of ferric ions when the pH of the acid is from 3 to 6 but is less than enough to precipitate a significant amount of calcium citrate. The amount of the 5-sulfosalicylic acid may be from 0.01 to 0.05 moles/l and the amount of citric acid is from 0.001 to 0.009 moles/l. 11 claims.

  19. Carnitine transport and fatty acid oxidation.

    Science.gov (United States)

    Longo, Nicola; Frigeni, Marta; Pasquali, Marzia

    2016-10-01

    Carnitine is essential for the transfer of long-chain fatty acids across the inner mitochondrial membrane for subsequent β-oxidation. It can be synthesized by the body or assumed with the diet from meat and dairy products. Defects in carnitine biosynthesis do not routinely result in low plasma carnitine levels. Carnitine is accumulated by the cells and retained by kidneys using OCTN2, a high affinity organic cation transporter specific for carnitine. Defects in the OCTN2 carnitine transporter results in autosomal recessive primary carnitine deficiency characterized by decreased intracellular carnitine accumulation, increased losses of carnitine in the urine, and low serum carnitine levels. Patients can present early in life with hypoketotic hypoglycemia and hepatic encephalopathy, or later in life with skeletal and cardiac myopathy or sudden death from cardiac arrhythmia, usually triggered by fasting or catabolic state. This disease responds to oral carnitine that, in pharmacological doses, enters cells using the amino acid transporter B(0,+). Primary carnitine deficiency can be suspected from the clinical presentation or identified by low levels of free carnitine (C0) in the newborn screening. Some adult patients have been diagnosed following the birth of an unaffected child with very low carnitine levels in the newborn screening. The diagnosis is confirmed by measuring low carnitine uptake in the patients' fibroblasts or by DNA sequencing of the SLC22A5 gene encoding the OCTN2 carnitine transporter. Some mutations are specific for certain ethnic backgrounds, but the majority are private and identified only in individual families. Although the genotype usually does not correlate with metabolic or cardiac involvement in primary carnitine deficiency, patients presenting as adults tend to have at least one missense mutation retaining residual activity. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler

  20. Ibotenic acid and thioibotenic acid

    DEFF Research Database (Denmark)

    Hermit, Mette B; Greenwood, Jeremy R; Nielsen, Birgitte

    2004-01-01

    In this study, we have determined and compared the pharmacological profiles of ibotenic acid and its isothiazole analogue thioibotenic acid at native rat ionotropic glutamate (iGlu) receptors and at recombinant rat metabotropic glutamate (mGlu) receptors expressed in mammalian cell lines....... Thioibotenic acid has a distinct pharmacological profile at group III mGlu receptors compared with the closely structurally related ibotenic acid; the former is a potent (low microm) agonist, whereas the latter is inactive. By comparing the conformational energy profiles of ibotenic and thioibotenic acid...... with the conformations preferred by the ligands upon docking to mGlu1 and models of the other mGlu subtypes, we propose that unlike other subtypes, group III mGlu receptor binding sites require a ligand conformation at an energy level which is prohibitively expensive for ibotenic acid, but not for thioibotenic acid...

  1. Agp2, a Member of the Yeast Amino Acid Permease Family, Positively Regulates Polyamine Transport at the Transcriptional Level

    KAUST Repository

    Aouida, Mustapha

    2013-06-03

    Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3? nor sam3? single mutant is defective in polyamine transport, while the dur3? sam3? double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2? mutant. Studies of Agp2 localization indicate that in the double mutant dur3? sam3?, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport. © 2013 Aouida et al.

  2. Agp2, a Member of the Yeast Amino Acid Permease Family, Positively Regulates Polyamine Transport at the Transcriptional Level

    KAUST Repository

    Aouida, Mustapha; Texeira, Marta Rubio; Thevelein, Johan M.; Poulin, Richard; Ramotar, Dindial

    2013-01-01

    Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3? nor sam3? single mutant is defective in polyamine transport, while the dur3? sam3? double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2? mutant. Studies of Agp2 localization indicate that in the double mutant dur3? sam3?, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport. © 2013 Aouida et al.

  3. Displacement of specific serotonin and lysergic acid diethylamide binding by Ergalgin, a new antiserotonin drug

    International Nuclear Information System (INIS)

    Oelszner, W.

    1980-01-01

    [ 3 H]-serotonin and [ 3 H]-lysergic acid diethylamide (LSD) bind with a high affinity, Ksub(D) = 12 nM and 6 nM, respectively, to distinct receptors of rat caudate membranes in vitro. Displacement experiments with unlabeled serotonin and LSD support the hypothesis of serotonin receptors existing in an agonist and antagonist state. Methysergide and Ergalgin display quite similar potenties in displacing [ 3 H]-serontonin and [ 3 H]-LSD from their specific binding sites (Ksub(i) = 46.7 and 53.4 nM; 22.3 and 36.5 nM, respectively). Contrary to pharmacological findings these binding results are in favour of mixed agonist/antagonist properties of these compounds. (author)

  4. Predicting HLA class I non-permissive amino acid residues substitutions.

    Directory of Open Access Journals (Sweden)

    T Andrew Binkowski

    Full Text Available Prediction of peptide binding to human leukocyte antigen (HLA molecules is essential to a wide range of clinical entities from vaccine design to stem cell transplant compatibility. Here we present a new structure-based methodology that applies robust computational tools to model peptide-HLA (p-HLA binding interactions. The method leverages the structural conservation observed in p-HLA complexes to significantly reduce the search space and calculate the system's binding free energy. This approach is benchmarked against existing p-HLA complexes and the prediction performance is measured against a library of experimentally validated peptides. The effect on binding activity across a large set of high-affinity peptides is used to investigate amino acid mismatches reported as high-risk factors in hematopoietic stem cell transplantation.

  5. Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles

    DEFF Research Database (Denmark)

    Norregaard, Kamilla; Andersson, Magnus; Nielsen, Peter Eigil

    2014-01-01

    This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation...... of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti......-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less...

  6. Gamma-hydroxybutyric acid (GHB) and the mesoaccumbens reward circuit: evidence for GABA(B) receptor-mediated effects.

    Science.gov (United States)

    Pistis, M; Muntoni, A L; Pillolla, G; Perra, S; Cignarella, G; Melis, M; Gessa, G L

    2005-01-01

    Gamma-hydroxybutyric acid (GHB) is a short-chain fatty acid naturally occurring in the mammalian brain, which recently emerged as a major recreational drug of abuse. GHB has multiple neuronal mechanisms including activation of both the GABA(B) receptor, and a distinct GHB-specific receptor. This complex GHB-GABA(B) receptor interaction is probably responsible for the multifaceted pharmacological, behavioral and toxicological profile of GHB. Drugs of abuse exert remarkably similar effects upon reward-related circuits, in particular the mesolimbic dopaminergic system and the nucleus accumbens (NAc). We used single unit recordings in vivo from urethane-anesthetized rats to characterize the effects of GHB on evoked firing in NAc "shell" neurons and on spontaneous activity of antidromically identified dopamine (DA) cells located in the ventral tegmental area. GHB was studied in comparison with the GABA(B) receptor agonist baclofen and antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH50911). Additionally, we utilized a GHB analog, gamma-(p-methoxybenzil)-gamma-hydroxybutyric acid (NCS-435), devoid of GABA(B) binding properties, but with high affinity for specific GHB binding sites. In common with other drugs of abuse, GHB depressed firing in NAc neurons evoked by the stimulation of the basolateral amygdala. On DA neurons, GHB exerted heterogeneous effects, which were correlated to the baseline firing rate of the cells but led to a moderate stimulation of the DA system. All GHB actions were mediated by GABA(B) receptors, since they were blocked by SCH50911 and were not mimicked by NCS-435. Our study indicates that the electrophysiological profile of GHB is close to typical drugs of abuse: both inhibition of NAc neurons and moderate to strong stimulation of DA transmission are distinctive features of diverse classes of abused drugs. Moreover, it is concluded that addictive and rewarding properties of GHB do not necessarily involve a putative high affinity GHB

  7. Evolution of sequence-defined highly functionalized nucleic acid polymers

    Science.gov (United States)

    Chen, Zhen; Lichtor, Phillip A.; Berliner, Adrian P.; Chen, Jonathan C.; Liu, David R.

    2018-03-01

    The evolution of sequence-defined synthetic polymers made of building blocks beyond those compatible with polymerase enzymes or the ribosome has the potential to generate new classes of receptors, catalysts and materials. Here we describe a ligase-mediated DNA-templated polymerization and in vitro selection system to evolve highly functionalized nucleic acid polymers (HFNAPs) made from 32 building blocks that contain eight chemically diverse side chains on a DNA backbone. Through iterated cycles of polymer translation, selection and reverse translation, we discovered HFNAPs that bind proprotein convertase subtilisin/kexin type 9 (PCSK9) and interleukin-6, two protein targets implicated in human diseases. Mutation and reselection of an active PCSK9-binding polymer yielded evolved polymers with high affinity (KD = 3 nM). This evolved polymer potently inhibited the binding between PCSK9 and the low-density lipoprotein receptor. Structure-activity relationship studies revealed that specific side chains at defined positions in the polymers are required for binding to their respective targets. Our findings expand the chemical space of evolvable polymers to include densely functionalized nucleic acids with diverse, researcher-defined chemical repertoires.

  8. Formic acid

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, H; Laing, B

    1921-12-03

    The production of formic acid by the oxidation of methane with a metallic oxide or by the interaction of carbon monoxide and water vapor in the presence of a catalyst, preferably a metallic oxide, is described along with the destructive distillation of carbonaceous material in the preesnce of formic acid vapor for the purpose of increasing the yield of condensible hydrocarbons.

  9. Adsorption of imidazolinone herbicides on smectite-humic acid and smectite-ferrihydrite associations.

    Science.gov (United States)

    Leone, P; Nègre, M; Gennari, M; Boero, V; Celis, R; Cornejo, J

    2002-01-16

    Adsorption of imazapyr (IMZ), imazethapyr (IMZT), and imazaquin (IMZQ) was studied on two smectite-humic acid and two smectite-ferrihydrite binary systems prepared by treating a Wyoming smectite with a humic acid extracted from soil (4 and 8% w/w of the smectite) and with just-precipitated synthetic ferrihydrite (8 and 16% w/w of the smectite). Adsorption of the three herbicides on the smectite was not measurable at pH >4.5, presumably because of negative charges on the surface of the smectite. Adsorption on the smectite-humic acid systems was also not measurable, presumably because of negative charges on the surface, despite the high affinity of the three herbicides for humic acid, the adsorption order of which was IMZ smectite-ferrihydrite systems and IMZQ smectite cannot adsorb herbicides, it modifies the adsorption capacity of ferrihydrite. The mutual interaction of active phases such as humic acid, ferrihydrite, and smectite alters the characteristics of the resulting surface and hence the adsorption process. Investigations of herbicide adsorption have been seen to produce more reliable results if conducted on polyphasic systems rather than on single soil components.

  10. Once-yearly zoledronic acid in the prevention of osteoporotic bone fractures in postmenopausal women

    Directory of Open Access Journals (Sweden)

    Irene Lambrinoudaki

    2008-09-01

    Full Text Available Irene Lambrinoudaki, Sophia Vlachou, Fotini Galapi, Dimitra Papadimitriou, K Papadias2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieio Hospital, GreeceAbstract: Zoledronic acid is a nitrogen-containing, third-generation bisphosphonate that has recently been approved for the treatment of postmenopausal osteoporosis as an annual intravenous infusion. Zoledronic acid is an antiresorptive agent which has a high affinity for mineralized bone and especially for sites of high bone turnover. Zoledronic acid is excreted by the kidney without further metabolism. Zoledronic acid administered as a 5 mg intravenous infusion annually increases bone mineral density in the lumbar spine and femoral neck by 6.7% and 5.1% respectively and reduces the incidence of new vertebral and hip fractures by 70% and 41% respectively in postmenopausal women with osteoporosis. Most common side effects are post-dose fever, flu-like symptoms, myalgia, arthralgia, and headache which usually occur in the first 3 days after infusion and are self-limited. Rare adverse effects include renal dysfunction, hypocalcemia, atrial fibrillation, and osteonecrosis of the jaw.Keywords: zoledronic acid, postmenopausal osteoporosis, bisphosphonate

  11. Structural and binding properties of two paralogous fatty acid binding proteins of Taenia solium metacestode.

    Directory of Open Access Journals (Sweden)

    Seon-Hee Kim

    Full Text Available BACKGROUND: Fatty acid (FA binding proteins (FABPs of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs of Taenia solium metacestode (TsM, a causative agent of neurocysticercosis (NC, shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2, which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1 and 8.4 (TsMFABP2. Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]aminoundecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions

  12. Effects of sarcoptic mange and its control with oil of Cedrus deodara, Pongamia glabra, Jatropha curcas and benzyl benzoate, both with and without ascorbic acid on growing sheep: assessment of weight gain, liver function, nutrient digestibility, wool production and meat quality.

    Science.gov (United States)

    Dimri, U; Sharma, M C

    2004-03-01

    The aim of this study was to evaluate the therapeutic efficacy of commonly used acaricidal drugs in India and also to assess the effect of ascorbic acid as adjunct therapy in 72 growing sheep with sarcoptic mange, aged 5-6 months and weighing 20.4-31.7 kg. Eight replicates of nine animals were formed based on sex, and day 0 body weight. Drugs were applied locally on the affected parts daily and recovery changes in skin lesions were observed at the time of every application. L-ascorbic acid was administered intramuscularly. Skin scrapings were collected daily from each group and examined for the presence of mites. Body weights were measured every 10th day from day 0 to 60. Nutrient digestiblity was evaluated by studying digestibility coefficients for dry matter, crude protein, ether extract, crude fibre, nitrogen free extract, total carbohydrates and nutrient balance (nitrogen, calcium and phosphorus) for a 30-day period. The liver function was evaluated by bromosulphophthalein (BSP) dye retention time. The animals were shorn on day 60 post-treatment (PT). Meat quality assesment was carried out by killing sheep at 60 days PT and estimating pH, water-holding capacity (WHC), tenderness, muscle colour, rib eye area and fat thickness. The lambs treated with oil of Jatropha curcas ascorbic acid had significantly (P retention time in sheep with sarcoptic mange. Post -treatment, the BSP retention time decreased in all treated groups and the decrease was maximum in OJC-AA treated group. The carcasses of sheep treated with OJC-AA had significantly (P water holding capacity, rib eye area and back fat thickness than the untreated infected control group. The muscle pH and tenderness values were significantly lower in OJC-AA treated group post-slaughter than infested untreated control group. Muscle colour of OJC-AA treated group was maximum bright red. The lambs treated with OJC-AA had significantly (P meat production.

  13. The role of G-protein-coupled receptors in mediating the effect of fatty acids on inflammation and insulin sensitivity.

    Science.gov (United States)

    Oh, Da Young; Lagakos, William S

    2011-07-01

    Chronic activation of inflammatory pathways mediates the pathogenesis of insulin resistance, and the macrophage/adipocyte nexus provides a key mechanism underlying decreased insulin sensitivity. Free fatty acids are important in the pathogenesis of insulin resistance, although their precise mechanisms of action have yet to be fully elucidated. Recently, a family of G-protein-coupled receptors has been identified that exhibits high affinity for fatty acids. This review summarizes recent findings on six of these receptors, their ligands, and their potential physiological functions in vivo. Upon activation, the free fatty acid receptors affect inflammation, glucose metabolism, and insulin sensitivity. Genetic deletion of GPR40 and GPR41, receptors for long-chain and short-chain fatty acids, respectively, results in resistance to diet-induced obesity. Deletion of GPR43 and GPR84 exacerbates inflammation, and deletion of the long-chain fatty acid receptors GPR119 and GPR120 reduces or is predicted to reduce glucose tolerance. These studies provide a new understanding of the general biology of gastric motility and also shed valuable insight into some potentially beneficial therapeutic targets. Furthermore, highly selective agonists or antagonists for the free fatty acid receptors have been developed and look promising for treating various metabolic diseases.

  14. A new boronic acid fluorescent sensor based on fluorene for monosaccharides at physiological pH.

    Science.gov (United States)

    Hosseinzadeh, Rahman; Mohadjerani, Maryam; Pooryousef, Mona; Eslami, Abbas; Emami, Saeed

    2015-06-05

    Fluorescent boronic acids are very useful fluorescent sensor for detection of biologically important saccharides. Herein we synthesized a new fluorene-based fluorescent boronic acid that shows significant fluorescence changes upon addition of saccharides at physiological pH. Upon addition of fructose, sorbitol, glucose, galactose, ribose, and maltose at different concentration to the solution of 7-(dimethylamino)-9,9-dimethyl-9H-fluoren-2-yl-2-boronic acid (7-DMAFBA, 1), significant decreases in fluorescent intensity were observed. It was found that this boronic acid has high affinity (K(a)=3582.88 M(-1)) and selectivity for fructose over glucose at pH=7.4. The sensor 1 showed a linear response toward d-fructose in the concentrations ranging from 2.5×10(-5) to 4×10(-4) mol L(-1) with the detection limit of 1.3×10(-5) mol L(-1). Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Cyclic phosphopeptides to rationalize the role of phosphoamino acids in uranyl binding to biological targets

    Energy Technology Data Exchange (ETDEWEB)

    Starck, Matthieu; Laporte, Fanny A.; Oros, Stephane; Sisommay, Nathalie; Gathu, Vicky; Lebrun, Colette; Delangle, Pascale [INAC/SyMMES, UMR5819, Universite Grenoble Alpes, CEA, CNRS, Grenoble (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' orme des Merisiers, Saint-Aubin, Gif-sur-Yvette (France); Creff, Gaelle; Den Auwer, Christophe [Institut de Chimie de Nice, UMR7272, Universite Cote d' Azur, Nice (France); Roques, Jerome [Institut de Physique Nucleaire d' Orsay, CNRS-IN2P3, Univ. Paris-Sud, Universite Paris-Saclay (France)

    2017-04-19

    The specific molecular interactions responsible for uranium toxicity are not yet understood. The uranyl binding sites in high-affinity target proteins have not been identified yet and the involvement of phosphoamino acids is still an important question. Short cyclic peptide sequences, with three glutamic acids and one phosphoamino acid, are used as simple models to mimic metal binding sites in phosphoproteins and to help understand the mechanisms involved in uranium toxicity. A combination of peptide design and synthesis, analytical chemistry, extended X-ray absorption fine structure (EXAFS) spectroscopy, and DFT calculations demonstrates the involvement of the phosphate group in the uranyl coordination sphere together with the three carboxylates of the glutamate moieties. The affinity constants measured with a reliable analytical competitive approach at physiological pH are significantly enhanced owing to the presence of the phosphorous moiety. These findings corroborate the importance of phosphoamino acids in uranyl binding in proteins and the relevance of considering phosphoproteins as potential uranyl targets in vivo. (copyright 2017 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim)

  16. Relationship between binding activity of sup 67 Ga and low sulfated acid glycosaminoglycans

    Energy Technology Data Exchange (ETDEWEB)

    Ohkubo, Yasuhito; Tsukada, Fumitake; Kohno, Hiroyuki (Tohoku Coll. of Pharmacy, Sendai (Japan)); Kubodera, Akiko (Science Univ. of Tokyo (Japan). School of Pharmaceutical Sciences)

    1989-01-01

    Sulfate content of acid glycosaminoglycan (AGAG) extracted from granuloma which had been produced by turpentine oil was inversely proportional to the amount of {sub 67}Ga accumulation in the granuloma. Additionally, the lowest sulfation occurred in granuloma at a peak of inflammation when the uptake of {sub 67}Ga had reached a maximum. On the basis of electrophoretic pattern, sulfate content, and specific optical rotation, it was concluded that acid glycosaminoglycans obtained from granuloma are mainly composed of chondroitin sulfate-A, -B, and desulfated heparin, while haparan sulfate was a minor component. From in vitro assays, desulfated acid glycosaminoglycans, especially desulfated-heparin and desulfated-heparan sulfate, were found to have a high affinity to {sub 67}Ga. These results suggest that low- or de-sulfation of AGAG is related to the accumulation of {sub 67}Ga in inflammatory lesions such as granuloma. Moreover, these results suggest that {sub 67}Ga does not bind to glycosaminoglycans via sulfuric acid residues. (author).

  17. Synthesis and characterization of in situ carbon-coated Li2FeSiO4 cathode materials for lithium ion battery

    International Nuclear Information System (INIS)

    Yan Zipeng; Cai Shu; Miao Lijuan; Zhou Xing; Zhao Yongming

    2012-01-01

    Highlights: → Sucrose and L-ascorbic acid as carbon additives. → Li 2 FeSiO 4 /C were obtained under our carbon coating experimental conditions. → Graphite-rich carbon was formed uniformly when ascorbic acid was employed. → The composites employing L-ascorbic acid exhibited a large specific surface area. → Sample using L-ascorbic acid exhibited better rate performance and cycling stability. - Abstract: Li 2 FeSiO 4 /C composites with in situ carbon coating were synthesized via sol-gel method based on acid-catalyzed hydrolysis/condensation of tetraethoxysilane (TEOS) with sucrose and L-ascorbic acid as carbon additives, respectively. As-obtained Li 2 FeSiO 4 /C composites prepared with L-ascorbic acid as a carbon additive are composed of nanoparticulate Li 2 FeSiO 4 in an intimate contact with a continuous thin layer of residual carbon and exhibit large specific surface area up to 395.7 m 2 g -1 . The results indicate that structure of the residual carbon is graphene-rich with obviously lower disordered/graphene (D/G) ratio. These as-obtained Li 2 FeSiO 4 /C composites exhibit first discharge capacity of 135.3 mAh g -1 at C/16 and perform cycling stability, which are superior to those of Li 2 FeSiO 4 /C composites synthesized with sucrose as a carbon additive.

  18. The Saccharomyces cerevisiae RAD18 gene encodes a protein that contains potential zinc finger domains for nucleic acid binding and a putative nucleotide binding sequence

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.S.; Prakash, L. (Univ. of Rochester School of Medicine, NY (USA)); Weber, S. (Kodak Research Park, Rochester, NY (USA))

    1988-07-25

    The RAD18 gene of Saccharomyces cerevisiae is required for postreplication repair of UV damaged DNA. The authors have isolated the RAD18 gene, determined its nucleotide sequence and examined if deletion mutations of this gene show different or more pronounced phenotypic effects than the previously described point mutations. The RAD18 gene open reading frame encodes a protein of 487 amino acids, with a calculated molecular weight of 55,512. The RAD18 protein contains three potential zinc finger domains for nucleic acid binding, and a putative nucleotide binding sequence that is present in many proteins that bind and hydrolyze ATP. The DNA binding and nucleotide binding activities could enable the RAD18 protein to bind damaged sites in the template DNA with high affinity. Alternatively, or in addition, RAD18 protein may be a transcriptional regulator. The RAD18 deletion mutation resembles the previously described point mutations in its effects on viability, DNA repair, UV mutagenesis, and sporulation.

  19. Preparation and characterization of fluorophenylboronic acid-functionalized affinity monolithic columns for the selective enrichment of cis-diol-containing biomolecules.

    Science.gov (United States)

    Li, Qianjin; Liu, Zhen

    2015-01-01

    Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity monolithic column applying 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) as a ligand. The DFFPBA-functionalized monolithic column not only exhibited an ultrahigh boronate affinity toward cis-diol-containing biomolecules, but also showed great potential for the selective enrichment of cis-diol-containing biomolecules in real samples.

  20. Lipoic Acid

    Directory of Open Access Journals (Sweden)

    Ramazan Tetikcok

    2015-09-01

    Full Text Available Lipoic acid, which is defined as a miralce antioxidan, is used by many departments. Eventhough clinical using data are very limited , it is used in treatment of diabetic neuropathy, physical therapy and rehabilitation clinic, dermatology clinic, geriatric clinics. It has usage area for cosmetic purposes. Although there are reports there are the direction of the effectiveness in these areas, the works done are not enough. Today lipoic acid , used in many areas ,is evaluated as universal antioxidant [J Contemp Med 2015; 5(3.000: 206-209

  1. Retention data of bile acids and their oxo derivatives in characterization of pharmacokinetic properties and in silico ADME modeling.

    Science.gov (United States)

    Trifunović, Jovana; Borčić, Vladan; Vukmirović, Saša; Kon, Svetlana Goločorbin; Mikov, Momir

    2016-09-20

    Information on ADME properties of examined bile acids and their oxo derivatives are scarce, although the interest for bile acids and their use in nanochemistry and macromolecular chemistry is increasing. The purpose of this research was to evaluate the lipophilicity, a crucial physicochemical parameter for describing ADME properties of selected bile acids and their oxo derivatives, and to compare two approaches: experimentally determined hydrophobicity parameters and calculated logP values. Commercially available bile acids - deoxycholic, chenodeoxycholic, hyodeoxycholic and ursodeoxycholic acid were used to synthesize oxo derivatives. Lipophilicity was evaluated in two solvent systems: toluene/ethanol and toluene/butanol. Retention parameters were acquired by normal-phase TLC. The correlations between calculated logP values obtained using five different software and experimentally determined hydrophobicity parameters (RM(0)(tol/eth), RM(0)(tol/but), b(tol/eth) and b(tol/but)) were examined. Correlation analysis confirmed significant dependence between experimental RM(0) values and software calculated parameters. Results suggest satisfactory intestinal absorption after oral administration for all of the examined compounds as well as low volumes of distribution, and high affinity for binding with plasma proteins. Penetration through blood-brain barrier and skin is not satisfactory. All of the examined compounds show high affinity for binding with G-protein coupled receptors and consequently inhibition of ionic channels. Results also suggest possible binding with nuclear receptors. Established lipophilicity testing model of studied compounds showed excellent predictive ability and might represent significant tool in development of relations between chromatographic behavior and ADME properties. Compounds 3α-hydroxy-7,12-dioxo-5β-cholanoic and 12α-hydroxy-3,7-dioxo-5β-cholanoic acid might be the most suitable candidates for further development studies (satisfactory

  2. Generation of Internal-Image Functional Aptamers of Okadaic Acid via Magnetic-Bead SELEX

    Directory of Open Access Journals (Sweden)

    Chao Lin

    2015-12-01

    Full Text Available Okadaic acid (OA is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb. Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA analysis, four candidate aptamers (O24, O31, O39, O40 were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 108 M−1, 1.47 × 109 M−1, 1.23 × 109 M−1 and 1.05 × 109 M−1, respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X − 1.78. The IC50 of O31 was 3.39 ng·mL−1, which was close to the value predicted by the OA ELISA (IC50 = 4.4 ng·mL−1; the IC10 was 0.33 ng·mL−1. The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay.

  3. A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples.

    Science.gov (United States)

    Kavita, Uma; Duo, Jia; Crawford, Sean M; Liu, Rong; Valcin, Joan; Gleason, Carol; Dong, Huijin; Gadkari, Snaehal; Dodge, Robert W; Pillutla, Renuka C; DeSilva, Binodh S

    2017-09-01

    We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and

  4. Carrier-mediated ¿-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

    2012-01-01

    and the anticancer prodrug d-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least...... two transporters seem to be involved in the basolateral transport of GABA. The basolateral uptake consisted of a high-affinity system with a K(m) of 290µM and V(max) of 75pmolcm(-2)min(-1) and a low affinity system with a K(m) of approximately 64mM and V(max) of 1.6nmolcm(-2)min(-1). The high...

  5. Dissolved Divalent Metal and pH Effects on Amino Acid Polymerization: A Thermodynamic Evaluation.

    Science.gov (United States)

    Kitadai, Norio

    2017-03-01

    Polymerization of amino acids is a fundamentally important step for the chemical evolution of life. Nevertheless, its response to changing environmental conditions has not yet been well understood because of the lack of reliable quantitative information. For thermodynamics, detailed prediction over diverse combinations of temperature and pH has been made only for a few amino acid-peptide systems. This study used recently reported thermodynamic dataset for the polymerization of the simplest amino acid "glycine (Gly)" to its short peptides (di-glycine and tri-glycine) to examine chemical and structural characteristics of amino acids and peptides that control the temperature and pH dependence of polymerization. Results showed that the dependency is strongly controlled by the intramolecular distance between the amino and carboxyl groups in an amino acid structure, although the side-chain group role is minor. The polymerization behavior of Gly reported earlier in the literature is therefore expected to be a typical feature for those of α-amino acids. Equilibrium calculations were conducted to examine effects of dissolved metals as a function of pH on the monomer-polymer equilibria of Gly. Results showed that metals shift the equilibria toward the monomer side, particularly at neutral and alkaline pH. Metals that form weak interaction with Gly (e.g., Mg 2+ ) have no noticeable influence on the polymerization, although strong interaction engenders significant decrease of the equilibrium concentrations of Gly peptides. Considering chemical and structural characteristics of Gly and Gly peptides that control their interactions with metals, it can be expected that similar responses to the addition of metals are applicable in the polymerization of neutral α-amino acids. Neutral and alkaline aqueous environments with dissolved metals having high affinity with neutral α-amino acids (e.g., Cu 2+ ) are therefore not beneficial places for peptide bond formation on the primitive

  6. Lactobacillus plantarum lipoteichoic acid inhibits biofilm formation of Streptococcus mutans

    Science.gov (United States)

    Ahn, Ki Bum; Baik, Jung Eun; Park, Ok-Jin; Yun, Cheol-Heui

    2018-01-01

    Dental caries is a biofilm-dependent oral disease and Streptococcus mutans is the known primary etiologic agent of dental caries that initiates biofilm formation on tooth surfaces. Although some Lactobacillus strains inhibit biofilm formation of oral pathogenic bacteria, the molecular mechanisms by which lactobacilli inhibit bacterial biofilm formation are not clearly understood. In this study, we demonstrated that Lactobacillus plantarum lipoteichoic acid (Lp.LTA) inhibited the biofilm formation of S. mutans on polystyrene plates, hydroxyapatite discs, and dentin slices without affecting the bacterial growth. Lp.LTA interferes with sucrose decomposition of S. mutans required for the production of exopolysaccharide, which is a main component of biofilm. Lp.LTA also attenuated the biding of fluorescein isothiocyanate-conjugated dextran to S. mutans, which is known to have a high affinity to exopolysaccharide on S. mutans. Dealanylated Lp.LTA did not inhibit biofilm formation of S. mutans implying that D-alanine moieties in the Lp.LTA structure were crucial for inhibition. Collectively, these results suggest that Lp.LTA attenuates S. mutans biofilm formation and could be used to develop effective anticaries agents. PMID:29420616

  7. Mefenamic Acid

    Science.gov (United States)

    Mefenamic acid comes as a capsule to take by mouth. It is usually taken with food every 6 hours as needed for up to 1 week. Follow ... pain vomit that is bloody or looks like coffee grounds black, tarry, or bloody stools slowed breathing ...

  8. Acid rain. Les pluies acides

    Energy Technology Data Exchange (ETDEWEB)

    Curren, T

    1979-11-28

    This report was produced for the use of Members of Parliament and House of Commons committees. The document describes the formation of acid rain, emissions of acidifying pollutants in North America, the growth of the problem and its environmental effects on aquatic and terrestrial ecosystems, human health and man-made structures. Areas of Canada which are most susceptible are identified. Actions taken by Parliament are given, including the formation of a sub-committee on acid rain and the passing of Bill C-51 in 1980 to amend the Clean Air Act, bringing it closer to a similar law in the U.S. A chronology of government responses to acid rain at the international, national and provincial level, is given. The most recent government actions included the passing of the US Clean Air Act by the Senate, the amending of the act into law, and commencement of negotiations to develop a Canada-US Air Quality Accord. 10 refs.

  9. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid

    Energy Technology Data Exchange (ETDEWEB)

    Hirose, Mika; Sugiyama, Shigeru, E-mail: sugiyama@chem.eng.osaka-u.ac.jp [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Ishida, Hanako; Niiyama, Mayumi [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Matsuoka, Daisuke; Hara, Toshiaki [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Mizohata, Eiichi [Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Murakami, Satoshi [Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Kanagaw 226-8501 (Japan); Inoue, Tsuyoshi [Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Matsuoka, Shigeru; Murata, Michio [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan)

    2013-11-01

    The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.

  11. Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid

    International Nuclear Information System (INIS)

    Hirose, Mika; Sugiyama, Shigeru; Ishida, Hanako; Niiyama, Mayumi; Matsuoka, Daisuke; Hara, Toshiaki; Mizohata, Eiichi; Murakami, Satoshi; Inoue, Tsuyoshi; Matsuoka, Shigeru; Murata, Michio

    2013-01-01

    The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS

  12. Nucleic acids encoding a cellulose binding domain

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1996-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  13. Levulinic acid

    Directory of Open Access Journals (Sweden)

    Barbara Hachuła

    2013-09-01

    Full Text Available The title compound (systematic name: 4-oxopentanoic acid, C5H8O3, is close to planar (r.m.s. deviation = 0.0762 Å. In the crystal, the molecules interact via O—H...O hydrogen bonds in which the hydroxy O atoms act as donors and the ketone O atoms in adjacent molecules as acceptors, forming C(7 chains along [20-1].

  14. Triptycene: A Nucleic Acid Three-Way Junction Binder Scaffold

    Science.gov (United States)

    Yoon, Ina

    intermediates, and provided a new regioisomer that was not observed in the previous synthesis. Through this investigation, we discovered new high-affinity lead compounds towards a d(CAG)·(CTG) trinucleotide repeat junction. In addition, we turned our attention to sigma 32 mRNA, which contains a RNA three-way junction in E. coli. We demonstrated that triptycene-based small molecules can modulate the heat shock response in E. coli..

  15. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    Directory of Open Access Journals (Sweden)

    Mashiur Rahman

    2014-12-01

    Full Text Available Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA was studied by equilibrium dialysis method (ED at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 and diazepam as site-2 specific probe. Results: Different analysis of binding of metoprolol succinate to bovine serum albumin suggested two sets of association constants: high affinity association constant (k1 = 11.0 x 105 M-1 with low capacity (n1 = 2 and low affinity association (k2 = 4.0×105 M-1 constant with high capacity (n2 = 8 at pH 7.4 and 27°C. During concurrent administration of palmitic acid and metoprolol succinate in presence or absence of ranitidine or diazepam, it was found that palmitic acid displaced metoprolol succinate from its binding site on BSA resulting reduced binding of metoprolol succinate to BSA. The increment in free fraction of metoprolol succinate was from 26.27% to 55.08% upon the addition of increased concentration of palmitic acid at a concentration of 0×10-5 M to 16×10-5 M. In presence of ranitidine and diazepam, palmitic acid further increases the free fraction of metoprolol succinate from 33.05% to 66.95% and 40.68% to 72.88%, respectively. Conclusion: This data provided the evidence of interaction at higher concentration of palmitic acid at the binding sites on BSA, which might change the pharmacokinetic properties of metoprolol succinate.

  16. Boronic acid based imprinted electrochemical sensor for rutin recognition and detection.

    Science.gov (United States)

    Wang, Chunlei; Wang, Qi; Zhong, Min; Kan, Xianwen

    2016-10-21

    Multi-walled carbon nanotubes (MWNTs) and boronic acid based molecular imprinting polymer (MIP) were successively modified on a glassy carbon electrode surface to fabricate a novel electrochemical sensor for rutin recognition and detection. 3-Aminophenylboronic acid (APBA) was chosen as a monomer for the electropolymerization of MIP film in the presence of rutin. In addition to the imprinted cavities in MIP film to complement the template molecule in shape and functional groups, the high affinity between the boronic acid group of APBA and vicinal diols of rutin also enhanced the selectivity of the sensor, which made the sensor display a good selectivity to rutin. Moreover, the modified MWNTs improved the sensitivity of the sensor for rutin detection. The mole ratios of rutin and APBA, electropolymerized scan cycles and rates, and pH value of the detection solution were optimized. Under optimal conditions, the sensor was used to detect rutin in a linear range from 4.0 × 10 -7 to 1.0 × 10 -5 mol L -1 with a detection limit of 1.1 × 10 -7 mol L -1 . The sensor has also been applied to assay rutin in tablets with satisfactory results.

  17. Binding of phenazinium dye safranin T to polyriboadenylic acid: spectroscopic and thermodynamic study.

    Directory of Open Access Journals (Sweden)

    Ankur Bikash Pradhan

    Full Text Available Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride with single and double stranded form of polyriboadenylic acid (hereafter poly-A using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A with high affinity while it does not interact at all with the double stranded (ds form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na⁺] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.

  18. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  19. High-affinity multivalent wheat germ agglutinin ligands by one-pot click reaction

    Directory of Open Access Journals (Sweden)

    Henning S. G. Beckmann

    2012-06-01

    Full Text Available A series of six mono-, di-, and trivalent N,N’-diacetylchitobiose derivatives was conveniently prepared by employing a one-pot procedure for Cu(II-catalyzed diazo transfer and Cu(I-catalyzed azide–alkyne cycloaddition (CuAAC starting from commercially available amines. These glycoclusters were probed for their binding potencies to the plant lectin wheat germ agglutinin (WGA from Triticum vulgaris by an enzyme-linked lectin assay (ELLA employing covalently immobilized N-acetylglucosamine (GlcNAc as a reference ligand. IC50 values were in the low micromolar/high nanomolar range, depending on the linker between the two disaccharides. Binding enhancements β up to 1000 for the divalent ligands and 2800 for a trivalent WGA ligand, compared to N,N’-diacetylchitobiose as the corresponding monovalent ligand, were observed. Molecular modeling studies, in which the chitobiose moieties were fitted into crystallographically determined binding sites of WGA, correlate the binding enhancements of the multivalent ligands with their ability to bind to the protein in a chelating mode. The best WGA ligand is a trivalent cluster with an IC50 value of 220 nM. Calculated per mol of contained chitobiose, this is the best WGA ligand known so far.

  20. High-affinity monoclonal antibodies specific for deoxynucleosides structurally modified by alkylating agents: Applications for immunoanalysis

    International Nuclear Information System (INIS)

    Adamkiewicz, J.; Ahrens, O.; Rajewsky, M.F.

    1984-01-01

    So far the results of attempts to use monoclonal antibodies for the demonstration of carcinogen-DNA adducts in cells by immunostaining have been promising. Thus the authors have established a standardized procedure for the quantitation of specific alkyl-deoxynucleosides in the nuclear DNA of individual cells by direct immunofluorescence, using tetramethylrhodamine isothiocyanate-labeled monoclonal antibodies and a computer-based image analysis of electronically intensified fluorescence signals. With a fluorescent anti-(O/sup 6/-EtdGuo) monoclonal antibody, the present detection limit for O/sup 6/-Etd-Guo in the nuclei of individual cells previously exposed to an ethylating N-nitroso compound (e.g., N-ethyl-N-nitrosourea) is -- 700 O/sup 6/-EtdGuo molecules per diploid genome, i.e., similar to the detection limit for the same ethylation product in a hydrolysate of (O/sup 6/-EtdGuo)-containing DNA analyzed by competitive RIA

  1. Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein

    DEFF Research Database (Denmark)

    Nielsen, Marianne Jensby; Petersen, Steen Vang; Jacobsen, Christian

    2006-01-01

    Haptoglobin-related protein (Hpr) is a primate-specific plasma protein associated with apolipoprotein L-I (apoL-I)-containing high-density lipoprotein (HDL) particles shown to be a part of the innate immune defense. Despite the assumption hitherto that Hpr does not bind to hemoglobin, the present...

  2. The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

    Energy Technology Data Exchange (ETDEWEB)

    Briers, Yves [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Schmelcher, Mathias; Loessner, Martin J. [Institute of Food Science and Nutrition, ETH Zuerich, Schmelzbergstrasse 7, CH-8092 Zuerich (Switzerland); Hendrix, Jelle; Engelborghs, Yves [Laboratory of Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200G, B-3001 Leuven (Belgium); Volckaert, Guido [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Lavigne, Rob, E-mail: rob.lavigne@biw.kuleuven.be [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium)

    2009-05-29

    The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD{sub KZ}), originating from Pseudomonas aeruginosa bacteriophage {phi}KZ, has been examined using a fusion protein of PBD{sub KZ} and green fluorescent protein (PBD{sub KZ}-GFP). A fluorescence recovery after photobleaching analysis of bound PBD{sub KZ}-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10{sup 7} M{sup -1} for the PBD{sub KZ}-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD{sub KZ}-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

  3. High Affinity, Developability and Functional Size: The Holy Grail of Combinatorial Antibody Library Generation

    Directory of Open Access Journals (Sweden)

    Kathrin Tissot

    2011-05-01

    Full Text Available Since the initial description of phage display technology for the generation of human antibodies, a variety of selection methods has been developed. The most critical parameter for all in vitro-based approaches is the quality of the antibody library. Concurrent evolution of the libraries has allowed display and selection technologies to reveal their full potential. They come in different flavors, from naïve to fully synthetic and differ in terms of size, quality, method of preparation, framework and CDR composition. Early on, the focus has mainly been on affinities and thus on library size and diversity. Subsequently, the increased awareness of developability and cost of goods as important success factors has spurred efforts to generate libraries with improved biophysical properties and favorable production characteristics. More recently a major focus on reduction of unwanted side effects through reduced immunogenicity and improved overall biophysical behavior has led to a re-evaluation of library design.